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Genetic Modification in Aquaculture A review of potential benefits and risks.

L. Galli Bureau of Rural Sciences

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© Commonwealth of Australia

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Galli, L. (2002). Genetic modification in aquaculture – A review of potential benefits and risks. Bureau of Rural Sciences, Canberra.

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Genetic Modification in Aquaculture 2

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Executive summary

This is an overview of current research into the use of modern biotechnology in aquaculture. It is directed to policy and decision makers to give an indication of issues relevant to research into and the potential for commercialisation of genetically modified (GM) organisms in the seafood industry.

The international aquaculture industry is exploring measures to increase their efficiency due to the growing demand for fish worldwide that cannot be met from wild-caught fish alone. Research into developing genetically modified fish is conducted in the United States of America, Canada, United Kingdom, New Zealand, Cuba, China, the Korean Republic and India. Some limited research is taking place in Australia. At present, no commercial use of transgenic fish has been approved anywhere in the world. On the other hand, the aquaculture industry is already taking advantage of efficiency gains from commercialisation of GM crops. Vegetable protein, an important component in aquaculture feed, is often derived from GM soybeans. Other feed ingredients could originate from GM crops as well.

Application of gene technology in fish to improve production efficiency has many potential benefits. Research on GM fish has primarily focused on producing fish with increased growth rates, increased temperature tolerance, and improved disease resistance. Fish have been modified to grow six times faster than normal, survive in colder climates, and possess natural disease resistance so important to high-density aquaculture.

Whilst the potential benefits of GM fish are plenty, there are some associated risks to consider prior to their use in commercial production. Ecological risks would arise if GM fish escaped from aquaculture facilities and into the wild. These genetically enhanced fish could potentially interact with the local wild population and produce reduced fitness, decline in other species in the community, transfer of disease and parasites, and a decrease in prey species. Preventative measures include sterilisation of all transgenic fish, and better aquaculture infrastructure to ensure secure containment of fish, neither of which yet is fully effective.

A number of potential human health risks have been suggested, however, there has been little evidence to date to indicate that GM fish are not safe for human consumption. Some consumers are still to be fully convinced. At the current level of public acceptance of GM food, the commercial risk would be quite high if GM fish were given the go ahead. This may change in the future as the potential benefits are further realised and potential ecological risks reduced. There has been some public reaction to the use of GM animal feed, particularly in some European countries. The same reaction has not yet been levelled at the aquaculture industry.

Genetic engineering researched…

… for use in aquaculture

Growth and survival improved …

… but potential threats to natural ecosystems …

… and human health must be considered

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Contents _______________________________________________________________________ Executive summary 3

Introduction 5

International research overview 7

Potential benefits 10

Increased growth 10 Growth Hormone 10 Sterilisation 12

Freeze resistance 12

Disease resistance 13

Feral pest control 13

Other 14

Future possibilities 14

Potential Risks 16

Ecological Risks 16 Intraspecific interactions 16 Introgression/displacement of population 18 Factors that reduce likely impact 19 Interspecific interactions 19 Transfer of disease and parasites 21 Measures to reduce environmental risks 21

Human health risks 23 Safety of food derived from gene technology 24

Commercial Risks 24

Risk Assessment 26

Conclusion 29

Acknowledgements 29

Glossary 30

References 34 Appendix 1: Producing GM fish 48

Microinjection 49 Electroporation 50 Triploidy 51 Others 51

Appendix 2: Transgene expression 52 Appendix 3: Definitions for risk assessment 53

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Introduction

Sixty to 70 percent of the world’s marine fisheries are threatened by over-fishing according to United Nations Food and Agricultural Organisation (FAO 2001). Aquaculture has been proposed as the only way to sustainably increase seafood production on a global scale. Agriculture, Fisheries and Forestry – Australia values Australia’s aquaculture industry at around $678 million, which comprises about 30% of the total value of fisheries production (AFFA 2001). The United Nations Food and Agricultural Organisation estimates that at some point between 2015 and 2025, half of all fish consumed in the world will be farmed (FAO 2001). Due to the rapidly increasing focus on aquaculture farming, the industry is continually looking at measures for improving efficiency and is starting to explore modern biotechnological avenues (Hew and Fletcher 2001). Various genetic technologies are emerging ranging from genetic modification to enhance growth efficiency, resistance to freezing and disease, to polyploidy manipulation to control reproduction.

Biotechnology is the term given to the range of agricultural, mechanical and industrial technologies that make use of the natural processes or products of living organisms. Modern biotechnology, often referred to as gene technology, is an extension of this. For the past 20 years, modern biotechnology has contributed to advances in biomedical sciences and pharmaceutical industries (Hew and Fletcher 2001). Gene technology covers techniques used to alter or move the genetic material of microorganisms, plants and animals, either within the organism or between different organisms. It allows genetic material to be transferred between completely unrelated species thus giving breeders greater options to incorporate characteristics into organisms that are not normally available to them. In traditional breeding programs only closely related species can be crossbred.

Gene technology has been applied extensively to improve agricultural plant production. Since the first worldwide release of commercial GM crops in 1996, such cropping has grown rapidly and now covers more than 70 different species according to Agriculture and Biotechnology Strategies (Canada) Inc. (ABS Canada 2001), grown by 5.5 million farmers over 52.6 million hectares (James, 2001). Two GM plants, cotton and carnation, have been approved for commercial release in Australia, each with two varieties. At the beginning of 2002, five GM crops with several different varieties, most produced overseas and imported to Australia, had been approved for inclusion in food products and some would also be used in animal feed including for aquaculture use.

Gene technology has also been utilised in research of genetically modified animals, including fish, the topic of this report. Most of the research undertaken on GM fish has been for aquaculture purposes and involves increasing growth rates. Australian scientists were among the first in the world to produce GM animals, but currently undertake little research on genetic modification of fish. There are currently no GM animals in commercial production in Australia according to the Office of the Gene Technology Regulator (OGTR 2001). Worldwide, most of the transgenic research on fishes is being conducted in the United States of America and Canada, while the United Kingdom, New Zealand, Cuba, China, the Korean republic and India were also involved in the year 2000 (FAO 2000). At present, the commercial use of transgenic fish is yet to begin with only one pre-market application submitted to U.S. Food and

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Drug Administration (FDA). There is no indication when and if FDA will approve the application but the company involved hopes for a commercial release in 2004. The uncertainty associated with the marketability of genetically modified organisms (GMOs) has been a significant barrier to investment in research and development. Both a Scottish and a New Zealand company abandoned their GM salmon research after unfavourable publicity, but a Canadian arm of a U.S. company is still pressing ahead (Reichhardt 2000). British scientists could be among the first to offer low-cost GM fish for commercial farming in the Far East, the Indian subcontinent and possibly Africa (Carrell 2001).

Whilst the potential benefits to industry are great, there are risks associated with the production and accidental release of transgenic fish. This report provides a summary of research on GM fish and other aquatic animals, discussing the various applications and their potential benefits and the associated potential ecological, human health and commercial risks.

Bureau of Rural Sciences 6

International research overview

Research on transgenic fish is currently underway for approximately 35 species worldwide (Table 1), including: trout, carp, catfish, tilapia, Pacific salmon such as chinook and coho, and various other members of the salmonid family (Reichhardt 2000). The Royal Society of Canada states that research on transgenic shellfish is less advanced than for transgenic finfish (RSC 2001). The first successful gene transfer in a bivalve mollusc was the introduction of retroviral vectors into the dwarf surf clam (Lu et al. 1996; RSC 2001). Considerable research has been undertaken in the Japanese abalone where growth hormones and other gene constructs have been introduced (RSC 2001). Table 1. Examples of genetically engineered aquatic animals. Modified from Royal Society of Canada (RSC 2001)

Species Latin name Reference

Abalone Haliotis diversicolor Sin et al. (1995) African catfish Clarias gariepinus Müller et al. (1992) Arctic char Salvelinus alpinus Pitkanen et al. (1999) Crustacea Artemia franciscana Gendreau et al. (1995) Atlantic salmon Salmo salar Fletcher et al. (1988) Blackhead seabream Acanthopagrus schlegli Sin (1997) Brown trout Salmo trutta Sin (1997) Channel catfish Ictalurus punctatus Dunham et al. (1987) Chinook salmon Oncorhynchus tshawytscha Devlin (1997) Coho salmon Oncorhynchus kisutch Devlin et al. (1997) Common carp Cyprinus carpio Chen et al. (1993) Cutthroat trout Oncorhynchus clarki Devlin (1997) Dwarf surfclam Mulinia lateralis Lu et al. (1996) Gilthead seabream Sparus auratus Knibb (1997) Goldfish Carasius auratus Zhu et al. (1985) Indian catfish Heteropneustes fossilis Sheela et al. (1990) Japanese medaka Oryzias latipes Inoue et al. (1990) Killifish Fundulus sp. Khoo (1995) Kuruma prawn Penaeus japonicus Preston et al. (2000) Largemouth bass Micropterus salmoides Goldberg (1998) Loach Misgurnus anguillicaudatus Zhu et al. (1986) Milkfish Chanos chanos Wu et al. (1998) Mud carp Cirrhinus chinensis MacLean et al. (1987) Mud loach Misgurnus mizolepsis Nam et al. (2000) Northern pike Esox lucius Gross et al. (1992) Rainbow trout Oncorhynchus mykiss Chourrout et al. (1986) Red crucian carp Cirrhinus auratus auratus Sin (1997) Silver crucian carp Cirrhinus auratus linda MacLean et al. (1987) Striped bass Morone americanus Goldberg (1998) Tilapia Oreochromis hornorum Guillen et al. (1998) Tilapia Oreochromis mossambicus Wu et al. (1998) Tilapia Oreochromis niloticus Brem et al. (1988) Walleye Stizostedion vitreum Khoo (1995) Wuchang bream Megalobrama amblycephala MacLean et al. (1987) Zebrafish Danio rerio Stuart et al. (1988)


There are two main techniques applied to produce transgenic fish, microinjection and electroporation, with the former being the most common and effective (see Appendix 1 for more information on production techniques). The highly technical nature of genetic engineering means that it can only be performed in a private biotechnology research laboratory, a government-run hatchery or a university. The animals produced in these labs may then be sold on to fish farmers where they can breed a stock of genetically altered fish.

Almost all of the research on transgenic fish worldwide has been for the aquaculture industry. Genetic modifications are being used to (Hew and Fletcher 2001):

• increase growth rates; • enable adaptation to extreme environments, such as through freeze/cold

resistance; • increase disease resistance; • control sexual maturation, fertility and sex differentiation; • enhance nutritional qualities and; • improve food utilisation.

GM fish are unlikely to play a primary role in the expansion of the aquaculture industry according to the Bureau of Rural Sciences. The technology, however, is likely to improve the profitability of aquaculture through reduced time to market and improved harvest quality (BRS 1998).

Internationally, geneticists at the University of Southampton in the UK believe they are close to proving that GM tilapia can be safely farmed without damaging ecosystems. The GM fish are being bred with growth hormone genes from chinook salmon causing these fish to grow three times faster than their natural counterparts. The British research is running in parallel with a major multinational European Commission funded research program, which is intended to override resistance from environmentalists and consumers by proving that GM fish are safe for the environment and human health. The University of Southampton researchers are planning to hold trials during early 2002 in Thailand to prove its GM tilapia are fully sterile by mixing them with wild fish in a special self-contained facility. The Southampton research team predicts their GM tilapia will be on sale in three to five years. Likewise, the Canadian company Aqua Bounty Farms, a subsidiary of a US company based in Massachusetts, is expected to get approval by 2004 from the US Food and Drug Administration to market fast-growing GM salmon. Canadian authorities still have to be convinced about the ecological impact of commercial production. The European Commission insists it has no plans to support the commercial farming of GM fish, claiming their research is to ensure European Union scientists can keep pace with global developments (Carrell 2001).

Only a small number of Australian laboratories are undertaking transgenic work with aquatic animals due to the expensive equipment required (Professor John Lucas, James Cook University, pers. comm). The main institution involved in gene technology research in fish species is CSIRO, with some other work being conducted by postgraduate students at universities.

CSIRO is currently undertaking a project which aims to develop a mechanism through which sterility can be induced transgenically to prevent escaped aquaculture species


from establishing feral populations in the wild. This ‘sterile feral’ technology is nearing commercial development and has the potential to provide a more secure playing field for the future application of genetic engineering in animal production. The technology involves inserting reconstructed elements of a species’ own genome into aquaculture broodstock. Three model organisms are currently being used in the sterile feral project: zebrafish, mice, and Pacific oysters. The technology is still undergoing development, and improvements will involve applying this technology to aquaculture species of interest and pest species such as carp, as well as parallel studies that examine the costs, benefits and risks surrounding the potential use of GMOs in food production.

Most of the biotechnology research in Australian aquaculture is focused on mapping the genome of commercially important species and developing molecular markers to identify useful genes (BRS 1998). This technology is being used for stock identification and breeding selection, to explore better aquaculture diets, and the detection of genetic markers for traits such as enhanced growth and disease resistance (CSIRO 2001; Hew and Fletcher 2001). Within Australia, this research is being applied to Black tiger prawns, Kuruma prawns, Pacific oysters, sea urchins (Thomas et al. 2000) and abalone. Gene technology is currently being used to explore gene function in these species not to genetically modify them (CSIRO 2001).

The production of triploid organisms for growth enhancement purposes is another area of Australian research. Triploids are not considered GMOs as they contain no novel genes, only an extra set of genes, and they can occur naturally, albeit rarely. Research is being conducted to develop and evaluate the effectiveness of polyploidy manipulation techniques on species such as Black tiger prawns and abalone (BRS 1998).


Potential benefits

Increased growth Growth Hormone Increasing the growth rate of fish by adding a growth hormone inducing gene is a common area of research for GM fish (Devlin et al. 1995; Devlin 1997). The commercial incentive for growth hormone (GH) technology is the opportunity to rear fish to market-sizes faster. The increase in growth rates achieved by genetic engineering is typically 200% to 600% depending on the species, the structure of the gene construct, and/or the nature of insertion. This rate greatly exceeds the 5% to 10% one-generation increases commonly achieved by artificial selection (Dunham et al. 1992; Zhu 1992; Chen et al. 1993; Devlin et al. 1994; Saunders et al. 1998). GH is normally produced only in the pituitary gland of animals, and it circulates at relatively low levels in the blood. Insertion of an extra GH gene broadens the range of tissues producing the hormone.

Various promoters are used in transgenic fish to drive growth hormone genes. A promoter is a sequence at the beginning of a gene that determines how often the gene is “switched on” to produce proteins – in this case growth hormone. Early experiments utilised human growth hormone attached to the metallothionein promoter from mice. Other promoters from viruses have also been used in transgenic fish (Muir and Howard, 1999). Recently some promoters from fish have been isolated and it is thought that the marketplace will better accept these than viral or rodent promoters (Rahman and Maclean 1999). Antifreeze protein (AFP) promoter genes are naturally occurring in some fish and have proven effective in driving expression of the GH gene in transgenic fish. GH inhibits AFP promoter genes from species such as flounder, but genes from the ocean pout are not affected (Li et al. 1985). Two different AFP-GH constructs have been created: AFP-GH chimeric gene construct and AFP-GHf with the AFP promoter linked to the chinook salmon GH cDNA and a mini GH gene respectively (Du et al. 1992). These two “all fish” constructs are used to generate GH transgenic fish (Hew and Fletcher 2001). Other fish promoters used include: trout and salmon metallothionein, carp B actin, salmon histone, and protamine from fish species.

When the AFP promoter gene from ocean pout is used it causes the GH transgene to be expressed most strongly in the liver of transgenic fish (Rahman and Maclean 1999) stimulating GH production in the liver and its signalling cascade that leads to increased growth rates. Although this new system has an inhibitory effect on normal pituitary function, the overall result is GH serum levels many times higher than normal.

There is some debate about whether transgenic fish grow to final adult sizes greater than those achieved by their non-transgenic siblings. Some researchers have found that transgenic Atlantic salmon grow faster initially, but do not achieve adult sizes larger than non-transgenic salmon (RSC 2001). Meanwhile, work on transgenic tilapia has produced fish that are more than 500g heavier than the largest size ever recorded for tilapia in the wild (Rahman and Maclean 1999). A summary of the various experiments and their success in increasing growth is given in Table 2. Muir and Howard (1999) have been experimenting with the gene for a salmon growth hormone


and found that it can make adult medaka grow up to 50% larger than normal. Increased levels of GH cause increased activity of insulin-like growth factor I (IGF-I), which is a serum protein produced by the liver and peripheral cells that stimulates cell division when it binds to a cell’s surface receptor. Cells affected include fibroblasts, prechondrocytes and other cells necessary for the development of new skeletal and cartilaginous tissue (Goodman 1993; RSC 2001).

Table 2: Growth enhancement in commercially important transgenic fish using GH genes (modified from Hew and Fletcher, 2001)

Fish Species Promoter Source of GH gene Growth increase

Reference

Common carp mMT RSV

Human GH gene Rainbow trout GH cDNA

10% 20-40%

Zhu et al. (1989) Zhang et al. (1990) and Chen et al. (1993)

Crucian carp mMT Human GH gene 70% Zhu (1992)

Catfish RSV Coho GH cDNA 20% Dunham et al (1992)

Loach mMT op-AFP

Human GH gene Chinook GH cDNA

100% 150%

Zhu et al. (1996) Tsai et al. (1995)

Tilapia CMV op-AFP

Tilapia GH cDNA Chinook GH cDNA

80% 100%

Martinez et al. (1996) Maclean et al. (1995)

Pike RSV BGH cDNA 012% Gross et al. 1992

Atlantic Salmon op-AFP Salmon GH cDNA GH minigene

200-900% 200-900%

Du et al. (1992a) Hew et al. (1995)

Pacific Salmon Op-AFP sockeye MT sockeye histone

Salmon GH cDNA Salmon GH gene Salmon GH gene

200-900% 500-1000%

Delvin et al. (1995) Delvin et al. (1994) Delvin et al. (1994)

Some studies have shown that high levels of GH may produce an inhibitory effect on growth (Zhang et al. 1990; Lu et al. 1992; Hernandez et al. 1997; Guillen et al. 1998; Martinex et al. 1999). In one study on tilapia, small differences in the amount of GH ectopically expressed in the transgenic animals produced big differences in the effect on growth. This suggests that, at least in this species, a very fine control operates at the level of growth regulation (Hernandez et al. 1997; de la Fuente et al. 1998; Martinez et al. 1999). Studies have shown that relatively weak promoters are more effective in enhancing growth (Hernandez et al. 1997; de la Fuente et al. 1998; Martinez et al. 1999) than strong promoters that show only modest levels of growth acceleration in transgenic carp and tilapia (Zhang et al. 1990; Chen et al. 1993; Hernandez et al. 1997).

Many studies have focused on salmon predominantly in Canada where GH genes (and occasionally insulin-like growth factor genes, Chen et al. 1995) attached either to antifreeze protein promoter (Du et al. 1992) or to metallothionein-B promoter (Devlin et al. 1994) have been used successfully to achieve large increases in salmonid growth rate (BRS 1998; Saunders et al. 1998; Stevens et al. 1998). GH genes from chinook salmon are often used (eg. Rahman and Maclean 1999). Other fish species have been used including Indian catfish (eg. India, Sheela et al. 1999), carp (eg. Israel, Hinits and Moav 1999), tilapia (eg. UK, Rahman and Maclean 1999, Germany, Brem et al.


1988), brown trout (eg. Sweden, Johnsson et al. 1996) and northern pike (eg. Gross et al. 1992).

Sterilisation The most widespread genetic manipulation in aquaculture worldwide is sterilisation, which is achieved through polyploidy manipulation. These techniques are used to induce animals to carry additional copies of their chromosomes, which results in sterility. The advantage of sterile organisms is that they use their energy to continually add to flesh, rather than seasonally shifting to the production of sperm and eggs. Growth rates can be greatly enhanced (BRS 1998). If fish eggs are subjected to a heat or pressure shock shortly after fertilisation, they retain an extra set of chromosomes, ending up with three sets rather than the normal two. The result is a triploid fish that does not develop normal sexual characteristics and the females are sterile (Reichhardt 2000). Triploid animals can occur rarely in nature. Triploids are not GMOs because no new genes are being introduced. Triploids are the only economically feasible method available for producing sterile fish destined for human consumption (O’Keefe and Benfey 1999).

The commercial and research applications of triploidisation are well documented (Thorgaard and Gall 1979; Thorgaard 1983; Utter et al. 1983; Chourrout et al. 1986; Don and Avtalion, 1986; Thorgaard 1986; Arai and Wilkins 1987; Kavumpurath and Pandian 1990; Varadaraj and Pandian 1990). Triploid oysters are available in Australia but the technology does not seem to have been widely adopted by the industry. Conversely the salmon industry has used triploid grower fish for some years (Thomas et al. 2000).

Another method of sterilisation is through blocking the release of gonadotropin. Gonadotropin releasing hormone (GnRH) is responsible for sexual maturation in fish, stimulating release of gonadotropins from the pituitary (Alestrom et al. 1992). cDNA sequences from a number of species have been isolated and cloned (Bond et al. 1991; Klungland et al. 1992). Scientists are looking at transgenic measures to sterilise fish by blocking the expression of the GnRH gene by antisense1 or ribozyme2 technology (Maclean 1998). Two European Commission research projects recently conducted were successful at producing a generation of transgenically sterile rainbow trout (Smith et al. 2001). The studies also showed that sterility could be reversed under controlled conditions through the injection of GnRH to fish at the time of sexual maturation. Thus, the inhibition of GnRH mRNA through antisense technology seems to be an effective method of inducing sterility in fish. However, the efficiency of this method is limited by the current success rate of transgenic technology for insertion of genes into fish genomes (Smith et al. 2001).

Sterilisation is also being looked at as a measure to reduce the ecological impacts of escaped transgenic and non-transgenic farmed fish into the wild (see section on ecological impacts).

Freeze resistance Increasing the temperature tolerance of fish would expand the options for aquaculture. A common gene transplant is that of antifreeze protein genes where the intent is to

1 Use of a complementary nucleotide sequence to inhibit expression of a gene.

2 An RNA molecule with catalytic activity


develop fish that have an increased adaptability (particularly salmonids) to very cold waters (Kapuscinski and Hallerman 1991; Maclean 1998). To avoid freezing, several fish species are able to produce antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) that can interact with ice crystals and effectively lower the freezing temperature (Ewart et al. 1999; Hew and Fletcher 2001). These proteins can also protect membranes from cold damage (Rubinsky et al. 1991; Hays et al. 1996; Tablin et al. 1996). Atlantic salmon cannot tolerate low temperatures due to the absence of the AFP or AFGP gene in its genome, which is a problem for sea pen culture in cold waters, eg. in the Northwest Atlantic. Therefore, there is great interest in developing a new strain of freeze tolerant salmon in these areas.

The relevant genes have been isolated from a number of different species, including winter flounder, sea raven and ocean pout. Studies have shown that purified AFP from the winter flounder injected into trout can provide effective protection against freezing temperatures (Fletcher et al. 1986). Similarly, Wang et al. (1995) found that transgenic goldfish harbouring an AFP gene from the ocean pout are significantly more cold tolerant than the non-transgenic control at 4°C. Wu et al. (1998) demonstrated similar results with juvenile tilapia. The AFP gene driven by its own promoter has been successfully transferred into Atlantic salmon and transmission to progeny was demonstrated (Shears et al. 1991; Fletcher et al.1992). However, whilst the transgenic fish expressed the transgene and did have detectable AFP in their blood, the amounts synthesised were too low to provide useful freeze resistance (Maclean 1998). Several new improvements are currently in development to enhance the antifreeze level in transgenic fish (Hew and Fletcher 2001).

Disease resistance The high densities in which fish are farmed make them susceptible to diseases caused by viruses, bacteria, fungi and protozoa. Improving the natural disease resistance of farmed fish would increase profitability (Fjalestad et al. 1993).

No gene transfers to resist disease and parasitism have yet been reported for fishes (Hew and Fletcher 2001). However, research is under way on the relevant major genes (Weatherly and Gill 1987a,b; Scrimshaw and Kerfoot 1987; Halstead 1988; Olivera et al. 1990; Kapuscinski and Hallerman 1991). One example is the antibacterial enzyme lysozyme (Maclean 1998). This enzyme is effective in the mucous of fish against a range of bacterial pathogens (Grinde 1989) and attempts to increase its concentration might prove beneficial.

Another avenue is the development of vaccines using gene technology. Recombinant DNA vaccines are being developed for infectious hematopoietic necrosis virus (IHNV), a fish rhabdovirus responsible for massive mortalities of chinook salmon and rainbow trout (Kapuscinski and Hallerman 1991).

Feral pest control Attempts have been made to control feral fish populations using gene technology. Grewe (1996) states that an inducible fatality gene (IFG) offers the possibility for long-term control of carp. The first step is finding the appropriate fatality gene. One option that has been considered is the protein ricin, which is found in the seed coats of the castor oil plant, Ricinus communis (Olsnes and Pihl 1982). The protein consists of two chains, alpha and beta. The alpha chain is cytotoxic and will cause death to the cell in which it is produced. The potential for using ricin in biological control would


thus comprise a gene coding for the alpha chain linked to an externally activated promoter in a transgenic construct. Only when externally activated would the toxin be produced and kill the cells in which it is formed. The benefit of this approach is that even if ingested, the dormant stage would not be fatal to other organisms due to the lack of activity.

After transferring the transgene construct into the carp genome, the next stages include the creation of a hatchery population that is homozygous for the transgene followed by the stocking of these fish into the wild. Grewe’s model showed that stocking rate and size/age of fish stocked were the most critical factors: “Achieving adult populations that are 50% transgenic requires many more than 50 generations at stocking rates less than 1% but only 1-2 generations at a stocking rate of 20%” (Grewe 1996). Once the pre-determined target introgression levels have been reached (eg. 40, 60 or 90%), the fatality gene will be activated. An example of a trigger is zinc-laden pellets, which would be added to a water body to activate the fatality gene and kill fish carrying the gene. Another potential trigger could be the onset of sexual maturity (Koehn et al. 2000).

Integrating a genetic weakness into wild populations via the stocking of transgenic fish containing an IFG appears to be a potential long-term strategy for controlling carp in Australian waters. However, further development is required to find the most appropriate inducible promoter and efficient delivery vector for the transgene. Integration into the wild populations would then depend on ecological variables and the efficiency of stocking strategies (Grewe 1996).

Other Other transgenic work on fish have studied gene regulation and function, developmental genetics, and the use of animals for production of human hormones such as insulin (RSC 2001).

Future possibilities Possible future applications include (Kinoshita et al. 1994; Maclean 1998; RSC 2001; Maclean and Penman 1990; Maclean, 1989; Chen and Powers, 1990):

• raising marine fish in fresh water; • manipulating the length of reproductive cycles; • increasing the tolerance of aquaculture species to wider ranges of environmental

conditions; • enhancing nutritional qualities and/or taste; • controlling sexual maturation to prevent carcass deterioration as fish age; • using transgenic fish as pollution monitors; • controlling sex differentiation and sterility; • creating fish that act as pollution monitors; • enabling fish to use plants as a source of protein; • using fish to produce pharmaceutical products; • "imprinting" fish with marker DNA sequences in order to facilitate population

studies in the wild; and


• improving host resistance to a variety of pathogens, such as Infectious Haematopoietic Necrosis Virus (IHNV), Bacterial Kidney Disease (BKD) and furunculosis.


Potential Risks

Ecological Risks The current facilities used in aquaculture farms do not ensure complete containment of stock, with many fish escaping from farms into the wild. If transgenic fish were bred in current aquaculture facilities, some fish would escape and interact with their wild counterparts and the rest of the aquatic community (RSC 2001). It is difficult to predict their impact on ecological systems because of experimental difficulties, although models can provide some information. Many of the studies have used predictive models, laboratory observations or have considered examples from related studies on the impacts of escaped farmed fish or other exotic species into natural systems. The latter can provide some understanding as to the potential impacts of transgenic fish on natural systems (Campton and Johnston 1985; Taggart and Ferguson 1986; Allendorf and Leary 1988; Guyomard et al. 1989; Hindar et al. 1991). However, no research and risk assessment, no matter how comprehensive in scope, will cover all possible outcomes of introducing transgenic fish into natural ecosystems (Kapuscinski and Hallerman 1991).

The scale and frequency of introductions of transgenic fish into a particular environment will greatly influence the degree of ecological risk involved (Kapuscinski and Hallerman 1991). The type and degree of ecological risk will vary depending on a number of factors. These are (RSC 2001; Kapuscinski and Hallerman 1991; Tiedje et al. 1989; Kapuscinski and Hallerman 1990a; Reichhardt 2000):

• the type of transgenic fish, namely the overall phenotypic effect of the transgene; • the adaptive ability of the transgenic animals to the local environment; • the fitness of the transgenic fish; • the health of local populations; • the normal ecological role of the host species (keystone species could have a

profound potential to impact ecosystems); • the potential for dispersal and persistence; and • the local environment itself.

The effects of escaped transgenic fish on wild ecosystems can be divided into two types: intraspecific (mainly ‘genetic’) and interspecific (mainly ‘ecological’) levels (Hindar et al. 1991; Krueger and May 1991). Other aspects such as the introduction of diseases and effects on population size may have an effect at both the inter- and intraspecific level (Hindar 1995).

Intraspecific interactions One of the biggest ecological risks associated with growing GM fish is their likely impacts on the native population if they escape from aquaculture facilities. If transgenic fish enter an ecosystem that contains the same species, the genetics of that population will change if they interbreed. The population will acquire a new gene or set of genes that could alter the fitness of that population (eg. reduced antipredator response – see below). Behaviours involved in reproduction, feeding, territorial defence, spatial or temporary habitat distributions, or other life history features could be affected leading to unpredictable changes in population dynamics and perhaps even destabilisation of the community (Kapuscinski and Hallerman 1990a; Kapuscinski


and Hallerman 1991; Gutrich and Whiteman 1998). Also, marine organisms, as compared to land mammals, have a great ability to disperse and expand their range due to their high mobility, so they can affect other populations beyond those in which they were originally introduced (Gutrich and Whiteman 1998).

The fitness of transgenic fish that have been introduced into natural settings will depend on the influence of transgenes on fitness related traits. Introduced transgenic fish that initially display low fitness may simply become extinct, or they may develop increased fitness in response to natural selection (Tave 1986; Kapuscinski and Hallerman 1991). Phenotypic changes to morphology, physiology and behaviour could theoretically have both positive and negative effects on fitness. Compounding this is the current inability to reliably predict the variation in phenotype that will be produced by insertion of any single gene construct (RSC 2001). Gene expression is often pleiotropic (ie. affects many traits) especially for genes of major effect (Falconer 1981; Knibb 1997). Thus, theoretically, changes from genetic modification may influence other traits in an accidental way (Palmiter and Brinster 1986; Purself et al.1989; Knibb 1997) creating further complications when trying to determine the potential ecological risks.

In the case of fish with antifreeze protein genes, the range of the transgenic fish would be potentially much greater than its parent (Bruggeman 1993). Antifreeze proteins would confer a selective advantage to salmon in any location where cold seawater currently constrains their life history. A smolt3 expressing antifreeze proteins would be able to emigrate at a smaller size and a younger age and gain weight (and fecundity) in the sea to spawn at a younger age than individuals of the local populations. Thus, transgenic Atlantic salmon may be able to out compete native populations (Hindar 1995).

Disease resistant GM fish could have some major intraspecific effects if they escape into wild populations. These disease resistant transgenic fish would be at a competitive advantage over native stock if a disease entered the region.

The number of accidentally released fish from aquaculture operations is considerable; in many spawning populations, released fish now outnumber wild fish (Gausen and Moen 1991). Below is some empirical evidence of interactions between cultured and wild fish:

• escaped farmed Atlantic salmon can spawn successfully in rivers in the North Atlantic and the Northeast Pacific;

• escaped farmed Atlantic and Pacific Salmon have destroyed the egg nests constructed by wild salmon;

• the breeding performance of farmed Atlantic salmon, particularly males, can be inferior to that of wild salmon; and

• as juveniles, the progeny of farmed Atlantic Salmon can compete successfully with, and potentially competitively displace, the progeny of wild Atlantic salmon (RSC 2001).

Therefore any changes to the genetics of farmed fish that increase their competitiveness needs to be carefully considered.

3 A smolt is a juvenile salmon capable of quickly adapting to transfer from fresh to salt water followed by a period of rapid growth.


Introgression/displacement of population One of the risks associated with the escape of transgenic fish into wild populations is the potential for transgenic fish to displace indigenous stocks. Transgenic organisms are capable of reproduction, and they have the potential to establish themselves in the environment as persistent populations, or to introduce transgenes into existing populations through introgression or other means (Bruggeman 1993). Much of this is reliant on the mating success rate of the transgenic fish and when it comes to GH fish, they might be at an advantage. A recent study conducted by Muir and Howard (1999) modelled the impact transgenic fish can have on a wild population’s viability. The model was created on the Japanese medaka to determine the role of size in mating success. Larger transgenic medaka males were found to have a 4-fold advantage over the smaller, non-transgenic males but were 30% more likely to die before reaching sexual maturity under aquarium conditions. These results, amongst others, were plugged into an experimental model to estimate what would occur if 60 transgenic males were introduced into a population of 60,000 wild medaka. It took only 40 generations for the GM fish to drive the population to extinction. This was called the “Trojan gene effect” (Muir and Howard, 1999). It is important to realise, however, that the prediction of extinction assumed that mature transgenic fish would be bigger than their wild counterparts – whereas the gene construct used (human GH) only increased the medaka’s juvenile growth rate, and produced adult fish no bigger than average. The viability of these fish was even worse - their survival to sexual maturity was reduced by as much as 78% compared with wild-type medaka, which suggests that they could wipe out a wild population very quickly (Muir and Howard, 1999).

In some parts of the world, introgressed and native populations coexist in the same regions (NB. introgression was caused by the escape of normal farmed fish, not GM fish), which causes concern that some native populations are in impending danger of loss through introgression or displacement (eg. Allendorf et al. 1980; Garcia-Marin et al. 1991; Hindar et al. 1991). A decline in local wild populations has been observed to accompany releases, in particular following disease introduction, introgression and/or displacement. In other cases, the reasons for the decline remain obscure, but it is possible that both competition between released and wild fish, and increased predation rates (including over harvesting) contributed to the decline (Hindar 1995). Conversely there have also been reports of no detectable introgression into indigenous populations despite substantial introductions from cultured stocks. Presumably the cultured fish have been unable to reproduce successfully in the new environments (eg. Wishard et al. 1984; Vuorinen and Berg 1989; Hindar et al. 1991).

It is important to recognise that many studies have observed that where there is a detectable effect in comparisons between indigenous and exogenous fish (including hybrids), indigenous fish appear to perform better (Hindar et al. 1991). Most local populations should be adapted to their particular environmental conditions, and so in the case of low-level releases, it is expected that natural selection will remove adverse laboratory genetic changes from wild populations (Vogt et al. 1985; Hindar et al. 1991; Knibb 1997). However, should the laboratory genetic changes be selected in the wild, then a potential risk exists even at very low levels of release (Knibb 1997). Unfortunately, little is known about the individual traits that contribute significantly to a species’ fitness or selective advantage (Bruggeman 1993). There are few practical options to reverse an increase in the transgenic population and this underlies much of


the concern over release into the environment of transgenic organisms (Knibb 1997). However, these events are problematic with all farmed fish, not just transgenic ones.

Factors that reduce likely impact Whilst there are some serious potential risks to native populations through the release of transgenic varieties, studies have reported that transgenic fish may not be fit enough to out compete their native equivalents. Some studies have shown that GH fish have a reduced antipredator response. In fish, antipredator responses have a genetic basis but are modified by learning (Magurran 1990). Predator-vulnerable risky phenotypes may not be selected against in farmed environments because the food supply is constant and predictable, and natural predators are lacking (see Kohane and Parsons 1989). Growth hormone increases the energy demand and thereby the feeding motivation of an animal, however it can also reduce the antipredator response as a result. One study on brown trout showed that both hatchery selection and GH injection consistently reduced antipredator behavioural responses in juveniles in the presence of a trout predator (Johnsson et al. 1996). Similar reports have been made on salmonids with hatchery-reared salmonids accepting higher predation risks during foraging than wild salmonids (Johnsson and Abrahams 1991; Johnsson et al. 1996; Abrahams and Sutterlin 1998; Abrahams and Sutterlin1999). This together with the observation that young transgenic salmon (Atlantic salmon) appear to have less effective camouflage, should mean they are less likely to survive in the wild (Devlin et al. 1994; Rahman and Maclean 1999).

In combination with the reduced predator avoidance behaviour, genetic engineering can affect the overall shape of transgenic fish, which can lead to a reduced swimming ability. A reduced swimming ability would be expected to increase vulnerability to predators. Swimming speeds of transgenic coho salmon has been reported as being significantly lower than those of non-transgenic controls of the same size and age (Devlin et al. 1999). Reduced swimming speeds may be caused by ontogenetic delay or from disruption of the locomotor muscles and/or their associated respiratory, circulatory and nervous systems (Farrell et al. 1997). However, others have reported no difference between the two groups (Hill et al. 2000). A reduction in swimming speed has not been observed in transgenic Atlantic salmon when compared to non-transgenic controls (Abraham and Sutterlin 1999; RSC 2001). In addition, Aqua Bounty Farms’ scientists have found that the GM Atlantic salmon seem more active than the wild-type fish. Another report states that transgenic emigrating smolts could potentially better avoid predation than wild smolts upon entering salt water, because they would adjust faster to the saline environment and thereby escape estuarine and coastal predation (Hindar 1995). These varying results may be as a consequence of the different species being studied or may depend on where exactly the extra genes are incorporated into the fish genome as they can exert subtly different effects (Reichhardt 2000). Thus, fish that exhibit reduced antipredator responses may not survive in wild environments and so cannot out compete their wild counterparts.

Interspecific interactions Another ecological risk associated with the escape of transgenic fish into wild populations is the potential impacts on the broader aquatic community. Released


transgenic fish stocks are thought to pose a risk to other species through niche4 expansion (Kapuscinski and Hallerman 1990a, 1991) and even speciation5 (Knibb 1997). Even if they do not spread their genes, transgenic fish could disrupt the ecology of streams by competing with native fish for resources (Hallerman and Kapuscinski 1992; Gutrich and Whiteman 1998; Reichhardt 2000; RSC 2001). Interspecific interactions would be in the form of competition for space, food and cover. Such interference competition is primarily mediated through aggressive behaviour towards other individuals. Size-related competitive ability of GH fish may be a mechanism by which it gains a competitive advantage over another species (Hindar 1995).

Cultured GH fish, if comparatively large, may prey upon smaller, wild fish (Gutrich and Whiteman 1998; RSC 2001). Escaped transgenic fish, which are larger than normal at a given age, may lead to increases in the size of their selected prey (Kapuscinski and Hallerman 1990a). They could also have bigger appetites (reviewed by Weatherly and Gill 1987; Kapuscinski and Hallerman 1991), which means they have the potential to alter the dynamics of other fish populations that are interconnected in the food web (Zalinskas and Balint 1998). Devlin et al. (1999) have found that GH transgenic coho salmon eat nearly three times as much food as their natural counterparts under laboratory conditions. Whether this would still be the case in the wild is uncertain (Reichhardt 2000).

Another concern is that changes in resource or substrate use might occur in transgenic fish as a result of their altered genes (Kapuscinski and Hallerman 1991; Gutrich and Whiteman 1998). A study by Johnsson et al. (1996) showed that resource allocation patterns in brown trout change rapidly as a consequence of hatchery selection, whilst this refers to non-transgenic farmed individuals, farmed transgenic individuals would most likely have similar differences in their resource allocation patterns. This means that other species that utilise the same resources as the “new” (transgenic) fish could compete with the transgenics for the shared resource. Introductions of exotic species like the brown trout (Salmo trutta) appear to be the major factor causing declines and disappearance of many native brook trout populations in eastern North America. Apparently, brown trout excludes brook trout from preferred feeding and resting positions in streams through interference competition (Kruegar and May 1991; Hindar 1995). A similar situation could occur with a transgenic fish that plays a novel ecological role.

Transgenic fish may pose greater ecological risks than escapes from conventional aquaculture because “organisms with novel combinations of traits are more likely to play novel ecological roles, on average, than are organisms produced by recombining genetic information existing within a single evolutionary lineage” (Tiedje et al. 1989; Bruggeman 1993). Transgenic salmonids with introduced antifreeze protein genes would definitely fall in the category of “ecological novelties”. For example, transgenic salmonids with introduced antifreeze protein genes could contribute to the northwards expansion of Atlantic salmon. This increase in distribution would put

4 A niche is the function or position of an organism or population within an ecological community. Also, the particular area within a habitat occupied by an organism. 5 Speciation is the evolutionary formation of new biological species, usually by the division of a single species into two or more genetically distinct ones.


Arctic char populations at risk, especially river populations which are considered to be less competitive in that environment than most other salmonids, and which therefore only thrive in physically extreme environments as in the northernmost rivers (Hindar 1995).

Gutrich and Whiteman (1998) suggest that the ecological risk posed by introduced GM fish in the marine environment would be relatively high compared to terrestrial and freshwater environments. GM aquatic organisms introduced into the ocean environment may have a relatively high rate of establishment because of the comparatively high fecundity of many marine organisms compared to those from freshwater environments. Also, marine life history strategies such as active settlement may enhance the probability of establishment.

Transfer of disease and parasites Diseases are a common and serious problem in aquaculture because of the large numbers of fish reared in confined areas. All organisms carry a full suite of viruses, bacteria and parasites, the composition of which is usually unique to each species. Plants and animals are carriers, reservoirs or vectors of disease within a region (Stewart 1991) and when they enter new regions they often bring these diseases with them. Cultured fish are quite often responsible for introducing pathogens and parasites into natural systems. In Norway, Atlantic salmon populations in 35 Norwegian rivers have been greatly reduced since 1975 following introductions of fish from hatcheries infected by the monogenean parasite Gyrodactylus salaris. Another example is furunculosis caused by the bacterium Aeromonas salmonicida. This pathogen was introduced to Norwegian fish farms with infected smolts from Scotland in 1985, and has spread to more than 65% of the total fish farms in Norway and 15% of the rivers by the end of 1991. This latter example shows the enormous potential that aquaculture activities have for the spread of diseases (Hindar 1995).

The transfer of disease and parasites from cultured fish to wild populations is a risk associated with aquaculture species in general and not a new phenomenon. However, where matters become further complicated is in the case of disease resistant GM fish. Whilst these fish are resistant to disease they may still act as reservoirs or vectors of viruses, bacteria and parasites. If these fish escape into wild populations, these pathogens may infect local populations causing them to decline whilst the transgenic individuals, because they are immune to the diseases these pathogens induce, may flourish.

Measures to reduce environmental risks Two options to reduce risks associated with GM fish have been proposed: complete physical containment of GM fish or the development of improved methods for biological containment of GM fish (Smith et al. 2001).

The likelihood of GM fish escaping into the wild can be reduced by moving marine based operations inland where containment can be much more readily achieved (Hindar et al. 1991). Entis (1997) states that by increasing the growth rate of fish, the costs of fish production are less, allowing for the cost-effective rearing of fish on inland sites using recycled water systems. Conversely, Smith et al. (2001) and Maclean and Laight (2000) state that complete physical containment of aquaculture species is not an economically viable option given the high cost of enclosed systems, particularly for sea-based farms. Contained recirculation systems may be a feasible


option in the future, however, the capital costs would be high (Maclean and Laight 2000).

The option of biological containment requires induction of sterility in transgenic individuals so they cannot breed successfully if they escape into the wild. If cultured fish can be made sterile, it would eliminate the potentially deleterious consequences of interbreeding between wild and cultured fish. Sterility can be induced through induction of triploidy shortly after fertilisation (eg. Chourrout 1987; Hindar et al. 1991) or through completely blocking the reproductive system at the level of the brain through treatment with gonadotropin-releasing hormone (GnRH) (Smith et al. 2001) (see section on sterilisation).

There are a number of reasons why triploidy is unlikely to be an effective mitigation tool in the near future. One reason is the cost associated with confirming sterility in fish before their transfer to net pens. It is unlikely the aquaculture industry would find it economically worthwhile (RSC 2001). A greater mortality and a higher incidence of morphological deformities make it even less advisable (RSC 2001). Also, existing technical limitations make it difficult to guarantee that every triploid individual will indeed be sterile (Thorgaard and Allen 1987; Kapuscinski and Hallerman 1990b). The time it would take to determine whether all of the escaped transgenic individuals were sterile would increase the risk of substantial or irreversible alterations (Hallerman and Kapuscinski 1992). Even then it would be virtually impossible to eradicate the remaining transgenic fish and restore ecosystems due to the lack of technical and financial resources (Kapuscinski and Hallerman 1991).

Transgenically induced sterility through inhibition of the production of GnRH seems to be the most viable method, however, the effectiveness of this technique is limited by the current state of technology in the field (Smith 2001). As for all introduced transgenes, the effect of a transgene integrated into the genome depends on the number of copies and the site of insertion.

It is important to recognise that whilst effective sterilisation may reduce the impacts on the wild stocks, it does not completely eliminate them. For example, sterile males may still be sexually active and therefore compete in courtship with fertile males (Hindar et al. 1991; RSC 2001). The development of the offspring of triploid males is severely impaired, resulting in death during the embryonic and larval stages. If the males are more successful on the whole than fertile males in acquiring mates (and this could very well be the case in particularly if females are more attracted to the larger males), then there is the potential that this may drive the population to extinction.

The potential problem of sterile males attempting courtship could be prevented through mandatory use of all female populations (Bye and Lincoln 1986; RSC 2001). It is possible to raise female salmon as fertile males by treating them with male sex hormones. So by using these “sex reversed’ males – which will be able to produce only female offspring - as breeding stock, and submitting a pressure shock to the eggs that they fertilise, it should be possible to raise GM fish that consist entirely of infertile females. However, much more empirical data are required before this measure could be deemed a viable risk aversion measure (Hindar et al. 1991).

Other concerns include the lack of knowledge of the behaviour of sterilised fish and the fact that escaped sterile fish still have the potential to spread pathogens. Organisms need not interbreed for there to be negative impacts on the environment, as


evident through the effects of exotic species introductions into wild ecosystems (RSC 2001).

Human health risks One of the major concerns by the public about GMOs is whether or not they are safe for human consumption. Many reports state that GM fish are as safe to eat as conventionally bred fish (Berkowitz and Kryspin-Sorensen 1994; Maclean and Laight 2000). Concerns may arise for two reasons, if the DNA is sourced from an allergenic protein or if the transgene causes an inactive toxin gene to be expressed (Berkowitz and Kryspin-Sorensen 1994). These dangers could be mitigated by a regulatory assessment procedure of the introduced gene on a case-by-case basis (Maclean and Laight, 2000), as is currently performed by government agencies with transgenic crops.

An allergenicity risk exists if the DNA is sourced from a protein that is known to cause an allergic reaction in some people. An example is transferring a shellfish protein to a teleost fish, which could cause an anaphylactic reaction in people allergic to shellfish (Berkowitz and Kryspin-Sorensen 1994).

Toxic effects may result from the insertion of a transgene into the host genome (Berkowitz and Kryspin-Sorensen 1994). Insertion of a transgene could possibly cause an inactive toxin gene to be expressed in a normally safe species of fish (RSC 2001). Occasionally, altering a gene in some way has additional, seemingly unrelated, effects on the phenotype. This phenomenon is known as pleiotropy. The Royal Society of Canada (RSC 2001) states that the development of transgenic fish might activate the expression of a gene that is not normally expressed, resulting in increased levels of a toxin. However, Berkowitz and Kryspin-Sorensen (1994) believe that the chances of such an event occurring are extremely small, as normally safe fish would be unlikely to contain toxin genes that can be activated. If unexpressed toxin genes did occur in these fish, toxic varieties of these fish would have already been discovered, as dormant toxin genes would be turned on by naturally occurring genetic events.

Some marine organisms, including some fish, are associated with powerful toxins (Lange 1990) but in well-known edible fish, these toxins are of exogenous origin and are not a product of the fish genome itself (Saito et al. 1984; Berkowitz and Kryspin-Sorensen 1994). The cause has always been the incursion of red tides, bacteria, or other causes external to the fish. None of the common food fishes are known to produce a toxin endogenous to the fish itself. Examples are the mullet and puffer fish which both contain toxins that are produced by marine bacteria that live in a symbiotic relationship with the fish (Tamplin 1990). However, the absence of toxin genes in all fish genomes is not a certainty as there are more than 20,000 species of teleosts alone (Berkowitz and Kryspin-Sorensen 1994).

There is some evidence available that suggests that fragments of dietary DNA can cross the intestine and enter tissues (Doerfler 2000). This process appears to be a natural occurrence at low levels. Most of the foreign DNA enters leucocytes, with some entering other cells such as hepatocytes. DNA fragments have also been shown to cross the placenta and enter foetal tissues, and in some cases, enter the chromosomes of individual cells (Doerfler 2000). McConnell (n.d) states that there is no evidence that these fragments of DNA represent functional genes and that


transgene material would only be a minute fraction of the ingested DNA. Doerfler (2000) further observed that DNA from food tends to disappear from white blood cells, spleen and liver cells within 24 hours, and thus being eliminated from the organism. For digested DNA to have any effect, the entire gene must pass through the intestinal wall intact, and then insert itself into the chromosome of an individual cell. Although viruses use this technique to great effect, the process has never been observed with digested DNA, let alone transgenic material.

There are some uneasiness about the use of viral promoters (such as CaMV) for transgene expression. To avoid the negative perceptions, promoters found in fish should be used to create ‘all fish’ genes (Rahman and Maclean 1999; Liu et al. 1990; Kryspin-Sorensen and Berkowitz 1993; Levy et al. 2000).

There are other issues not necessarily of health interest but more issues of consumer satisfaction. Increased growth rates caused by transgenesis may have an effect on meat and nutritional quality. GH coho salmon are different in their muscle architecture consistent with increased rates of hyperplasia. Changes in the levels of muscle enzymes PFK and Cytox affect metabolism, which can lead to changes in meat quality (RSC 2001). Changes in nutritional quality is less likely with Kryspin-Sorensen and Berkowitz (1993) stating that many vitamins and minerals in fish are obtained through the food chain (algae and phytoplankton), therefore transgenesis does not interfere with the nutritional value of the fish (Kryspin-Sorensen and Berkowitz 1993).

Safety of food derived from gene technology All GM food in Australia is now subject to safety assessments by the Australia New Zealand Food Authority (ANZFA) before being approved for sale. ANZFA is responsible for developing and reviewing Australian and New Zealand food standards. A new food standard (Australian Food Standards Code A18 or Standard 1.5.2 in the joint Australia New Zealand Food Standards Code) to regulate the sale of foods produced using gene technology, was incorporated into the Food Standards Code on 13 May 1999 and amended in December 2000. Labelling of food derived from gene technology has been required since December 2001. In addition, the Codex Alimentarius Commission assesses the potential risks associated with food and if necessary develops measures to manage these risks.

The safety of transgenic animals for food has been considered internationally by a joint consultation of the FAO and WHO (WHO 1991) and by the OECD (1992). They have concluded that modern techniques of molecular biology and biotechnology do not inherently result in foods that are less safe than their closest conventional counterparts (Levy et al. 2000).

Commercial Risks A primary issue of producing novel strains of transgenic fish is their eventual demand by the aquaculture industry and consumers. Public backlash may be so fierce that, even if transgenic fish are produced, they do not sell. Commercial risks include market access restrictions, price discounts and dominance of large multinational companies.

In the US, the award of the first patent for a GM animal promoted intense debate. Religious groups questioned the morality of genetic modification stating that moral, social and spiritual issues should be considered more seriously before binding


decisions are made on the issue. In addition, some animal rights groups also did not support genetic engineering stating that animals will physically suffer as a result of genes being transferred into their genetic code (Crawford 1987; Hallerman and Kapuscinski 1990a). The farming community surveyed were in two minds about transgenic animal patenting. Some farmers stated that patenting would be an incentive for the rapid development of improved transgenic strains of livestock, whilst others were concerned about the potential of having to pay royalties for transgenic animals and their offspring, as well as being required to license rather than own their livestock (Hallerman and Kapuscinski 1990a).

At present, the commercial use of transgenic fish has not yet started and it is difficult to gauge the likely public reaction. Both Otter Ferry Salmon in Scotland and the New Zealand King Salmon Company scrapped their GM salmon research after unfavourable publicity. But Aqua Bounty Farms, the Canadian arm of a company based in Massachusetts, are still pressing ahead (Reichhardt 2000). They are producing AquAdvantage fish by taking a gene promoter sequence and splicing it to the growth hormone expression system of the salmon. AquAdvantage salmon have year-round expression of their growth hormone rather than just seasonal. The result is fish that grow from 400% to 600% faster than standard fish during the first year of life. These fish do not grow larger than their standard counterparts. The rapid growth rate diminishes as the fish reach their natural adult size (Entis 1997). The South Korean government pre-empted any launch of GM fish products by requiring that they be labelled accordingly and threatening violators with large fines (Reuters Ltd 2001).


Risk Assessment

A number of potential hazards associated with GM fish have been identified in this report. In order to make some judgement about the seriousness of these issues, a qualitative risk assessment was undertaken to serve as a basis for discussion only. This assessment is not based on a formal impact assessment and should not be taken to apply to any specific situation. The risk assessment model in the FRDC-Ecological Sustainable Development case study project - Case Study Information Package (Fletcher et al. 2002) - was used as a basis for conducting this risk assessment.

Potential hazards were identified and judged according to the level of consequence and their likelihood of occurring (see Table 3 and Appendix 3). From these two figures, the overall risk level was categorised as shown in Table 3.

Table 3: Risk Descriptor Matrix

Likely 6 Negligible Low Moderate High Extreme Extreme

Occasional 5 Negligible Low Moderate High Extreme Extreme

Possible 4 Negligible Low Moderate Moderate High Extreme

Unlikely 3 Negligible Low Low Moderate Moderate High

Rare 2 Negligible Low Low Low Moderate Moderate

Lik

eli

ho

od

le

vel

Remote 1 Negligible Low Low Low Low Low

0 1 2 3 4 5

Negligible Minor Moderate Severe Major Catastrophic

C o n s e q u e n c e l e v e l Consequence levels were assessed using the definitions in the Fletcher et al. (2002) report. Likelihood and consequence levels are further defined in Appendix 3.

Each potential hazard was then ranked from the highest to the lowest according to the overall risk level within the particular risk category (ie ecological, human health and commercial).

The risk levels assigned to each potential hazard depend on a number of factors and therefore the overall risk level may vary somewhat depending on the specific circumstances. For example, the type of disease, the scale and frequency of GM fish introductions, the type of transgenic fish, etc are all important (see section on Ecological Risks – Description). Rankings can be used to determine which of the potential hazards should be considered first when determining measures for risk elimination or reduction of likelihood or consequence. Risks were assessed on the basis of current state of GM fish technology, knowledge of impacts, aquaculture management practices, health safety assessments, and public acceptance of GM food.


Table 4: Overall risk levels of GM fish potential impacts.

Potential consequence Level of consequence

Likelihood of occurring

Overall risk level

Ecological Decrease in prey species (caused by increased predation from GH fish)

Severe Possible Moderate

Extinction of entire population (see information on “Trojan gene effect” in section on Ecological Risks- Introgression/displacement of population)

Catastrophic Remote Low

Decline in other species in community through niche expansion (change in prey species, habitat preference etc) of transgenic fish or speciation.

Severe to Major Rare Low to Moderate

Introduction of diseases/parasites Minor to catastrophic Possible Low to Extreme

Reduced fitness of entire population due to introgression/displacement

Moderate to Severe Rare Low

Decline of competitive species in “new” locations (in case of freeze resistant fish which may have greater distribution than native stock).

Severe to Major Remote Low

Human health

Allergenicity Severe Rare to Remote Low

Toxicity Severe Remote Low

Reduction in quality Minor Possible Low

Commercial

Public do not accept GM fish and do not buy them.

Catastrophic Likely to occasional

Extreme

Too expensive to produce GM fish safely

Catastrophic Likely to occasional

Extreme

Market access restrictions Severe Possible Moderate

Dominance of large multinational companies.

Moderate Possible Moderate

Price discounts Minor Likely Low

It would appear that there are many potential ecological risks associated with GM fish and many of these would have a high consequence, however, the likelihood of these risks occurring is quite low. The risk level of disease/parasite introduction was the most difficult to determine as it depends on the type of disease (amongst other factors) and the level of consequence could vary from minor to catastrophic. This risk already exists with aquaculture species in general.

The overall levels of potential human health risks are relatively low. GM fish would not pose a serious risk to human health mainly because the likelihood of these risks


occurring is very low as food products are subjected to intensive food safety assessments.

Many of the commercial risks were assigned a high overall risk level. These ratings are due to the current perceived lack of public acceptance of GM products as well as the current capacity of the aquaculture industry to produce GM fish in an environmentally friendly and yet economically viable manner.


Conclusion

Gene technology can provide many potential benefits for the aquaculture industry, including increased growth rates, increased temperature tolerance, and improved disease resistance. It is anticipated that further interest will develop in the future for using this tool to improve economic efficiency for the aquaculture industry as well as reduce pressures on wild stocks. There are, however, some associated risks with the application of gene technology in aquaculture. The potential effects on wild ecosystems from escaped farmed transgenic fish, human health safety and the reputation and viability of industries adopting this practice all have to be considered. Therefore, before the application of gene technology in aquaculture can be readily endorsed, the potential risks need to be thoroughly assessed and the necessary risk aversion measures developed and applied.

Acknowledgements

I want to acknowledge the assistance of Dr Stefan Fabiansson, Project Manager for the Food and Gene Technology Unit within the Bureau of Rural Sciences for refining the final text of this report. I also want to thank the following people for reviewing this report and providing constructive comments: Sandy Thomas, Dr Jean Chesson, Dan Quinn, Dr Julie Glover and Alex McNee – Bureau of Rural Sciences, Dr Julian O’Dea – Agriculture, Fisheries and Forestry – Australia, and Dr Paul J. Verma – Adelaide University.


Glossary

Alleles: Alternative forms of a genetic locus; a single allele for each locus is inherited separately from each parent (eg. at a locus for eye colour the allele might result in blue or brown eyes). Allergen: Any substance which can induce an allergy, that is an abnormal immunological reaction to a substance. Antifreeze protein: Proteins found in the extracellular fluid of some fish that live in very cold water which inhibit the formation of ice crystals. Antisense: Of or relating to a nucleotide sequence that is complementary to a sequence of messenger RNA. When antisense DNA or RNA is added to a cell, it binds to a specific messenger RNA molecule and inactivates it. Bacteriophage: A virus which infects bacteria. Base sequence: The order of nucleotide bases in a DNA molecule. Biotechnology: Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use. cDNA (copy DNA): DNA synthesized from an RNA template using reverse transcriptase. Construct: An artificially assembled DNA segment prepared for transfer into a target tissue. Typically, the construct will include the gene of particular interest, a marker gene and appropriate control sequences. Chimeric DNA: A molecule of DNA that has resulted from recombination, or has resulted from DNA from two sources being spliced together. Chromosomes: The self-replicating genetic structures of cells containing the cellular DNA that bears in its nucleotide sequence the linear array of genes. In prokaryotes, chromosomal DNA is circular, and the entire genome is carried on one chromosome. Eukaryotic genomes consist of a number of chromosomes whose DNA is associated with different kinds of proteins. Cytotoxic: Something which kills cells. Cytoplasm: The substance of the body of a cell, as distinguished from the karyoplasm, or substance of the nucleus. DNA (deoxyribonucleic acid): The molecule that encodes genetic information. DNA is a double-stranded molecule held together by weak bonds between base pairs of nucleotides. The four nucleotides in DNA contain the bases: adenine (A), guanine (G), cytosine (C), and thymine (T). In nature, base pairs form only between A and T and between G and C; thus the base sequence of each single strand can be deduced from that of its partner. Ectopic: A biological event or process that occurs in an abnormal location or position within the body. Electroporation: The process where an electric pulse is used to push things across the barrier of the cell membrane. Endogenous: Developed or added from within the cell or organism. Enzymes: Proteins that act as catalysts, speeding the rate at which biochemical reactions proceed but not altering the direction or nature of the reactions. Exogenous: Produced outside of, originating from, or due to external causes. Opposite of endogenous. Expression (gene expression): The process by which a gene’s coded information is converted into the structures present and operating in the cell. Expressed genes


include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but not translated into protein (e.g., transfer and ribosomal RNAs). Fitness: Biology. The extent to which an organism is adapted to or able to produce offspring in a particular environment. Gamete: A reproductive cell having the haploid number of chromosomes, especially a mature sperm or egg capable of fusing with a gamete of the opposite sex to produce the fertilized egg. Gene: The fundamental physical and functional unit of heredity. A gene is an ordered sequence of nucleotides located in a particular position on a particular chromosome that encodes a specific functional product (i.e., a protein or RNA molecule). Gene technology: The processes which enable genes to be moved from one organism to another, often unrelated, organism. Genetic modification: The manipulation of an organism's genetic endowment by introducing or eliminating specific genes through modern molecular biology techniques. Genome: All the genetic material in the chromosomes of a particular organism; its size is generally given as its total number of base pairs. Genotype: The genetic constitution of an organism. Compare phenotype. Germline: A group of cells in most multicellular animals which give rise to the reproductive cells. The genome of the animal as contained in these cells, along with any mutations which might arise in them (germline mutations), can be passed on to offspring. Also can refer to the appearance and conditions of the genome in the germ cells which may be different from within the somatic cells. Also, the development of a germ cell as originating from a cell in a zygote. Glycoprotein: A protein linked to a sugar or polysaccharide which are components of receptor molecules on the outer surface of cells. Gonadotropin releasing hormone (GnRH): The hormone produced and released by the hypothalamus that controls the pituitary gland’s production and release of gonadotropins. Gonadotropins (gonadotropic hormones): Gonadotropins are the hormones produced by the pituitary gland that control reproductive function. They include follicle stimulating hormone (FSH) and luteinizing hormone (LH). Growth hormone: A hormone which stimulates the growth of bones and muscles in juvenile animals (including human children). It can also be artificially added to adult domestic animals to increase the growth of muscles or production of milk in adult animals. It is produced by the pituitary gland in the brain. Hepatocytes: A parenchymal cell of the liver Heterozygous: Containing two different alleles of the same gene. Homozygous: Containing two copies of the same allele. Hormone: A biochemical substance that is produced by a specific cell or tissue and causes a change or activity in a cell or tissue located elsewhere in an organism. Hyperplasia: An abnormal increase in the number of cells in an organ or a tissue with consequent enlargement. Interspecific: arising or occurring between members of different species. Intraspecific: arising or occurring within a species; involving the members of one species; "intraspecific competition".


Introgression: Infiltration of the genes of one species into the gene pool of another through repeated backcrossing of an interspecific hybrid with one of its parents. In vitro: In an artificial environment outside the living organism: an egg fertilized in vitro; in vitro fertilization. Locus: The position on a chromosome of a gene or other chromosome marker; also, the DNA at that position. The use of locus is sometimes restricted to mean regions of DNA that are expressed. Mapping (gene mapping): Determination of the relative positions of genes on a DNA molecule (chromosome or plasmid) and of the distance, in linkage units or physical units, between them. Marker: An identifiable physical location on a chromosome (e.g., restriction enzyme cutting site, gene) whose inheritance can be monitored. Markers can be expressed regions of DNA (genes) or some segment of DNA with no known coding function but whose pattern of inheritance can be determined. Microinjection: A technique for introducing a solution of DNA, protein or other soluble material into a cell using an extremely fine instrument. Mosaic: An organism or part of an organism that is composed of cells with different origin. Nucleotide: A subunit of DNA or RNA consisting of a nitrogenous base (adenine, guanine, thymine or cytosine in DNA; adenine, guanine, uracil, or cytosine in RNA), a phosphate molecule, and a sugar molecule (deoxyribose in DNA and ribose in RNA). Thousands of nucleotides are linked to form a DNA or RNA molecule. Pantropic: Having an affinity for or indiscriminately affecting many kinds of tissue: pantropic viruses.

Peptide: Any of various natural or synthetic compounds containing two or more amino acids linked by the carboxyl group of one amino acid to the amino group of another. Phenotype: The physical appearance/observable characteristics of an organism. Compare phenotype. Plasmid: A circular, double-stranded unit of DNA that replicates within a cell independently of the chromosomal DNA. Plasmids are most often found in bacteria and are used in recombinant DNA research to transfer genes between cells. Pleiotropism, pleiotropy: The control by a single gene of several distinct and seemingly unrelated phenotypic effects Polar body: A reproductive cell having the haploid number of chromosomes, especially a mature sperm or egg capable of fusing with a gamete of the opposite sex to produce the fertilized egg. Polyploid (polyploidy): Having more than the normal (diploid) number of chromosomes; a multiple of the haploid number that is caused by chromosomal replication without nuclear division. Promoter: A site on DNA to which RNA polymerase will bind and initiate transcription. Protein: A large molecule composed of one or more chains of amino acids in a specific order; the order is determined by the base sequence of nucleotides in the gene coding for the protein. Proteins are required for the structure, function, and regulation of the body cells, tissues, and organs, and each protein has unique functions. Examples are hormones, enzymes, and antibodies.


Provirus: The precursor or latent form of a virus that is capable of being integrated into the genetic material of a host cell and being replicated with it. Recombinant DNA: A combination of DNA molecules of different origin that are joined using recombinant DNA technologies. Restriction enzyme: Any of a group of enzymes that catalyse the cleavage of DNA at specific sites to produce discrete fragments, used especially in genetic engineering. Also called restriction endonuclease. Retrovirus: Any of a group of viruses, many of which produce tumours that contain RNA and reverse transcriptase, including the virus that causes AIDS.

Ribozyme (RNA enzyme): A molecule of ribonucleic acid that has catalytic activity.

RNA (ribonucleic acid): A chemical found in the nucleus and cytoplasm of cells; it plays an important role in protein synthesis and other chemical activities of the cell. The structure of RNA is similar to that of DNA. There are several classes of RNA molecules, including messenger RNA, transfer RNA, ribosomal RNA, and other small RNAs, each serving a different purpose.

Sequence: The noun: the order in which subunits appear in a chain, such as amino acids in a polypeptide or nucleotide bases in a DNA or RNA molecule. The verb: To find out in what order the subunits appear in the chain.

Speciation: The evolutionary formation of new biological species, usually by the division of a single species into two or more genetically distinct ones.

Stem cell: An unspecialised cell that gives rise to a specific specialised cell, such as a blood cell. Strain: A population of cells all descended from a single cell; also called a clone.

Transcription: The synthesis of an RNA copy from a sequence of DNA (a gene); the first step in gene expression.

Transfection: Infection of a cell with purified viral nucleic acid, resulting in subsequent replication of the virus in the cell.

Transgenic: This term describes an organism that has received sequences of DNA by artificial means, followed by integration of one or more of the novel sequences into their chromosomal DNA (Maclean and Leight 2000).

Translation (protein synthesis): The process in which the genetic code carried by messenger RNA directs the synthesis of proteins from amino acids.

Triploid: Having or being a chromosome number three times the haploid number.

Vector: An organism that spreads an infectious disease; often, this infectious host is not affected by the illness. Also, a DNA molecule that replicates on its own in a host cell and can be used as a vehicle in the laboratory for replicating other types of DNA. Definitions distilled from Dictionary.com - available at: www.dictionary.com.; Biotech’s Life Science Dictionary - available at: http://biotech.icmb.utexas.edu/search/dict-search.html.; and the FAO Glossary of Biotechnology and Genetic Engineering - available at: http://www.fao.org/DOCREP/003/X3910E/X3910E00.htm.


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Appendix 1: Producing GM fish

Transgenic animals were first produced in 1980 by direct microinjection of in vitro recombinant DNA (Brem et al. 1988). From a technical perspective, transgenic fish are easier to produce than transgenic mammals, as fish eggs undergo external fertilisation, therefore removing the problem of transfer to surrogate mothers (Kryspin-Sorensen and Berkowitz 1993). However, the usual method for mammals is in vitro cell transfection followed by cell selection, and there is no piscine equivalent of the mammalian embryonic stem cell system, therefore this method is not feasible for producing transgenic fish. Thus, there is no satisfactory method of gene targeting in fish. There is an advantage however. Cytoplasmic injection of the DNA is used instead and this method ensures a higher survival rate of the embryo than nuclear injection (Kryspin-Sorensen and Berkowitz 1993). There are two primary techniques used to produce transgenic fish, microinjection and electroporation. Microinjection is the most commonly applied method as it is the most effective.

TECHNIQUE - The gene that expresses the particular trait of interest (eg. faster growth, disease resistance) is

firstly isolated. This involves taking tissue from the animal that naturally has the desired gene, homogenising it and separating the nuclear DNA from mitochondrial DNA and other cell debris. The genomic DNA is then treated with a number of restriction nuclease enzymes so that a number of lengths of DNA result.

- Each of these lengths of DNA are then spliced into a vector - usually a plasmid which is a circular piece of DNA that can be reproduced inside bacteria (other type of vector can be a bacteriophage, which is a virus that infects bacteria eg. pantropic retroviral vector).

- The plasmids then go inside the bacteria and replicate. Billions of copies of the plasmid carrying the desired gene (the transgene) are produced as the bacteria grow in the culture.

- Once mass numbers of plasmids are produced, they are isolated from the bacteria. - The plasmid is then genetically edited to change its structure from circular into a linear bit of

DNA. In addition to the gene for the desired trait are other flanking nucleic acid sequences which co-ordinate the expression of the gene. One of these regulatory sequences is a promoter, it acts to switch on and off the transcription and translation of the foreign gene. A gene on its own does not work, it needs a promoter to be expressed.

- The plasmid containing the transgene is then either physically injected into a fertilized egg using a needle viewed under the microscope (called microinjection – see below) or through electroporation (see below).

- The plasmid carries the gene into the nucleus of the cell permanently integrating the gene into the chromosome. This process is technically demanding and it is difficult to control where in the chromosome the transgene is inserted, which sometimes causes variations in the level at which the gene is expressed.

- Once the eggs hatch, a heterogeneous population is produced in which some individuals are genetically changed and others are not. (Currently less than 5 percent of offspring that make it to birth will carry the new gene integrated in such a way that it actually functions).

- All individuals are screened to identify those fish that carry the transgene. To show that the material is part of a chromosome, a Southern blot has to be performed after the genomic DNA has been treated with restriction enzymes and run on an electrophoresis gel. By using restriction enzymes, it can be determined whether the new DNA is incorporated as a whole, only partly, or whether it appears to have been incorporated more than once in the genome of each cell.

- The next stage is to find out whether the gene is being expressed. This is usually done by detecting the protein end product and is usually done with antibodies to the protein. If the antibodies detect the protein in the blood and/or cell extracts of the individual, it is important to determine whether that material is derived from the recombinant genetic material or if it comes from native genes. If growth hormone levels (for example) are statistically significantly


higher in transgenic individuals compared to controls, the recombinant gene is probably being expressed.

- When the gene is integrated, inherited and expressed, the transgenic organisms acquire new genotypes and phenotypes depending on the nature of the gene introduced and the specificity and strength of the promoters driving the expression (Levy et al. 2000; Hew and Fletcher 2001).

- The transgenics are then used to create the breeding stock. The novel gene can be passed on vertically through to any offspring. This means that microinjection or electroporation may only have to be performed with one generation of animal. However, this is very dependent on how the novel gene is incorporated into the genome and how many copies are present.

- Recombinant integration may only be heterozygous, ie there may only be one copy of the foreign gene in the genome, thus only 50% of any gametes produced could carry the recombinant gene. If more than one copy of the recombinant gene is incorporated in the genome they may occur on different chromosomes, homozygously or heterozygously; the resulting transgenic ratio of offspring will be complex, but will be greater than 50%.

(Information distilled from Lewis 2001, Australian Biotechnology Association 2001, Deakin University).

Microinjection The most commonly applied method to introduce novel gene constructs into fish is microinjection of the transgene into the cytoplasm of the embryo (Hackett 1993; Maclean and Rahman 1994; Kang et al. 1999; Preston et al. 2000). The act of microinjection slightly reduces the chance of egg survival, as the large number of molecules required to increase the probability of integration may prove lethal to the developing embryo (Penman et al. 1990; Fletcher and Davies 1991). In addition, the eggs that do survive injection may or may not incorporate some of the DNA. There are two main factors that determine whether this occurs. Firstly, the number of copies of the gene that are injected (commonly between 1000 and 1000000) ─ because the injection is cytoplasmic, a large copy number is needed to offer a reasonable probability of integration of injected sequences into the nucleus (Maclean 1998) ─ and, secondly, how close to the egg nucleus the injection places the DNA.

The method of microinjection is time consuming, if not impractical for single-generation mass production due to the hard egg envelope and/or the opaqueness of the eggs in many species (Muller et al. 1992; Hackett 1993; Kang et al. 1999). However, it is much more efficient than any other method currently available, including electroporation. In one study on penaeids, approximately 500 times more plasmids recovered following microinjection compared with that recovered through electroporation. (Preston et al. 2000).

The problem of the tough egg envelope (otherwise called the chorion) has been tackled in various ways. Dechorionation by digestion and/or mechanical removal has been successfully used for species such as goldfish and loach, Misgurnus anguillicaudatus (Zhu et al. 1985) and zebrafish (Stuart et al. 1988). These species rapidly develop to hatching. Rainbow trout embryos can be successfully microinjected shortly after fertilisation, prior to the hardening of the envelope (Maclean and Talwar 1984), or through an approach where a hole is drilled in the envelope of each embryo through which the transgene is injected (Chourrout et al. 1986; McEvoy et al. 1988, 1992). Alternatively, glutathione can be used to prevent hardening of the envelope (Oshiro et al. 1989; Inoue et al. 1991).


Electroporation A less common method of introducing transgenes is electroporation (Pandian et al. 1999). Fertilized eggs and/or sperm are immersed in a solution containing foreign DNA and then subjected to a series of short electrical pulses to permeabilise cell membranes, thereby permitting the entry of DNA molecules into cells (Kang et al. 1999; Royal Society of Canada 2001). This procedure is relatively simple, as it requires less skill than microinjection. However, it has a low efficiency rate of gene transfer obtained with DNA incorporation rates ranging from 2.6% (Muller et al. 1992) to 50% (Tsai et al. 1995; Kang et al. 1999). Effective gene transfer by electroporation is dependent on a number of electrical and biological parameters (Andreason and Evans 1988). The osmotic condition of the sperm during electroporation, among other factors, is one parameter that has the potential to influence the efficiency of gene transfer (Kang et al. 1999). Others include field strength, the number of pulses applied, and DNA concentration (Symonds et al. 1994a; Sin et al. 1993; Symonds et al. 1994b; Sin et al. 1995; Tsai 2000).

Electroporation of fertilised eggs can produce 10 to 100 times greater numbers than can microinjection (Powers et al. 1992). However, gene transfer efficiency is still not high enough to handle the tremendously large number of eggs spawned within a very short time by aquacultural species (Tsai 2000). Successful attempts have been reported however (Khoo et al. 1992; Muller et al. 1992: Symonds et al. 1994a; Tsai 2000). Electroporation of embryos soon after fertilisation has been successfully used to transfer genes into medaka (Inoue et al. 1990) and zebrafish (Buono and Linser 1991; Powers et al. 1992) embryos (Tsai 2000). Tsai’s (2000) study showed that the GH gene transferred by electroporation was functional in transgenic finfish and shellfish and resulted in fast-growing transgenic varieties (Tsai 2000). Lu et al. (1996) used electroporation to successfully transfer a pantropic pseudotyped retroviral vector containing the envelope protein of vesicular stomatitis virus into fertilised eggs of the dwarf surfclam, Mulinia lateralis. Conversely, electroporation has been ineffective on goldfish embryos (Hallerman et al. 1989) and rainbow trout (Cherrout and Perrot 1992; Tsai 2000). Preston et al. (2000) found in their electroporation study on prawns that much of the DNA was bound to the exterior of the embryos.

Sperm cells can also be used as carriers for introducing foreign DNA into eggs, therefore permitting the mass production of transgenic animals Muller et al. (1992) attempted gene transfer by sperm electroporation in carp and reported gene transfer rates of 2.6-4.2% (Kang et al. 1999). This method would be particularly useful in transgenic studies on shellfish such as abalone. The low survival rate of the embryos of some shellfish species from pre-settlement (about 1-7%) (Tong and Moss 1992) makes microinjection of embryos unfeasible as a method for gene transfer. Thus, a mass gene transfer technique using sperm cells as a vector would be more appropriate because a large number of eggs can be treated efficiently (Sin et al. 1995). Powers et al. (1992) achieved an 83% rate of DNA transfer when both sperm and eggs of carp were electroporated. Whilst this result is impressive, sperm electroporation on its own is simpler and more practical for mass production, since a larger number of gametes can be treated at one time.

One problem with electroporation is the apparently low rate of integration of the introduced DNA into the host cells (Kang et al. 1999).


Triploidy Triploid individuals are mostly sterile with a reduced gonadal development, particularly in females (Purdom 1993). Triploids can occur spontaneously in nature, although they are very rare (Maclean and Laight 2000). The most commonly applied method of inducing triploidy is retention of the second polar body after the second meiotic division of the oocyte to produce the mature egg (Purdom 1970; Maclean and Laight 2000). Second polar body retention can be generated by temperature, pressure, or chemical treatment of the fertilised egg.

Triploid individuals possess three complete chromosome sets in their somatic cells and differ from conspecific diploids in three ways. They are more heterozygous, have larger although fewer cells in most tissues and organs, and their gonadal development is disrupted to some extent, depending on the sex, ie. females typically remain immature (Royal Society of Canada 2001).

Others Another less known technique is particle bombardment. This method was unsuccessful on prawns (Preston et al. 2000) but has been applied successfully to embryos of another marine crustacean (Artemia) (Gendreau et al. 1995) and in fish (Zelenin et al. 1991; Preston et al. 2000).

Gene constructs can also be introduced via transfection of novel genes into embryonic stem cells, followed by their reintroduction into the inner cell mass of the developing embryo. This technique is still in the early stages (RSC 2001).


Appendix 2: Transgene expression

The major problem with genetic engineering is expression of the gene, ie. transcription and translation. If this does not happen, the novel gene is of no use. In only a few cases has germline transmission and stable long term transgene expression been satisfactorily demonstrated in the several species of fish that have been used to produce lines of transgenic fish (Rahman and Maclean 1999). Studies have shown that transgene expression varies depending on which region of the host genome the transgene integrates into (position effect) (Lin et al. 1994; Martinez et al. 1999; Maclean and Laight 2000). Following injection into fish embryos, some of the transgenes are randomly integrated into the host genome, while others are degraded, resulting in the production of mosaic transgenic fish and low frequencies of germline transmission (Dunham et al. 1992; Maclean 1998; Martinez et al. 1999). Hence, the first progeny of the genetically altered fish are very heterogeneous and include non-transgenic fish and mosaic fish that differ from one another. To establish a family of transgenic fish, one of the transgenic individuals needs to be bred (Hinits and Moav 1999).

Another factor that reduces the level of transgene expression is the origin of the transgene construct used. Initially, scientists used promoter sequences and GH gene sequences from very distantly related species (Brem et al. 1988; Rokkones et al. 1989; Penman et al. 1990; Zhang et al. 1990). When transgene sequences of mammalian origin were used, irregular or nil expression of the transgene frequently resulted (Guyomard et al. 1989; Rokkones et al. 1989; Penman et al. 1990; Rahman and Maclean 1999). Wherever feasible, transgene constructs as near as possible to homologous both with respect to promoter and structural genes should be used, partly because they are found to be more effective (Alam et al. 1996), but also because of the probability that use of the subsequent fish in aquaculture would have increased customer acceptance. This is particularly true with respect to sequences of viral origins due to the public’s perception of the risks associated with the use of viral or bacterial sequences (eg. Martinez et al. 1996; Rahman and Maclean 1999).


Appendix 3: Definitions for risk assessment

Consequence definitions (From Fletcher et al. 2002)

Level Ecological

Negligible (0) General - Insignificant impacts to habitat or populations, Unlikely to be measurable against background variability

Target Stock/Non-retained: undetectable for this population

Ecosystem: Interactions may be occurring but it is unlikely that there would be any change outside of natural variation

Habitat: Affecting < 1% of area of habitat.

Minor (1) Target/Non-Retained: Possibly detectable but no impact on population size or dynamics.

Ecosystem: Captured species do not play a keystone role – only minor changes in relative abundance of other constituents.

Habitat: Possibly localised affects < 5% of total habitat area

Rapid recovery would occur if stopped - measured in days to months.

Moderate (2) Target: Full exploitation rate where long term recruitment/dynamics not adversely impacted

Non Retained:

Ecosystem: measurable changes to the ecosystem components without there being a major change in function. (no loss of components)

Habitat: 5-30 % of habitat area is affected.

:Or, if occurring over wider area, the impact to habitat from activity is not major

Recovery measured in months - years

Severe (3) Target/Non Retained: Affecting recruitment levels of stocks/ or their capacity to increase

Ecosystem: Ecosystem function altered measurably and some function or components are missing/declining/increasing outside of historical range &/or allowed/facilitated new species to appear.

Habitat: 30- 60 % of habitat is affected/removed.

Recovery measured in years.

Major (4) Target/Non Retained: Likely to cause local extinctions

Ecosystem: A major change to ecosystem structure and function (different dynamics now occur with different species/groups now the major targets of capture)

Habitat: 60 - 90% affected

Recovery period measured in years to decades.

Catastrophic (5) Target/Non Retained: Local extinctions are imminent/immediate

Ecosystem: Total collapse of ecosystem processes.

Habitat: > 90% affected in a major way/removed

Long-term recovery period will be greater than decades.


Likelihood Definitions

Level Description

Likely It is expected to occur in most circumstances

Occasional Will probably occur in most circumstances

Possible Might occur at some time

Unlikely Could occur at some time

Rare May occur in exceptional circumstances

Remote Never heard of, but could occur

Ranking of Risk Level

Risk Rankings Risk Values

Negligible 0

Low 1-6

Moderate 7-12

High 13-18

Extreme >19


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