Aquaculture 250 (2005) 542 – 554 www.elsevier.com/locate/aqua-online

Review

Introduction of genetic engineering in aquaculture: Ecological and ethical implications for science and governance Anne Ingeborg Myhra,T, Roy Ambli Dalmob a Norwegian Institute of Gene Ecology, The Science Park, P.O. Box 6418, N-9294 Tromsø, Norway Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway

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Received 9 March 2004; received in revised form 23 December 2004; accepted 26 December 2004

Abstract Within aquaculture, genetic engineering (GE) is emerging as a powerful method for breeding of fish and shellfish, and for developing alternative sources of feed and vaccines to combat diseases. On the other hand, the use of GE in aquaculture raises ecological, ethical and economic concerns. For instance, genetically modified (GM) feed could be spread to the aquatic environment and consumed by other marine organisms, and horizontal gene transfer may conceivably occur from DNA in feed or vaccines to a recipient genome or by faeces to the environment. Numerous reports have described beneficial effects such as viral disease resistance following DNA vaccination. However, side effects, such as activation of other genes than those which are central in immune defence mechanisms, may occur and warrant further investigations. In order to achieve sustainable introduction of GE, it is crucial that appropriate scientific investigations and ethical considerations are done prior to large-scale introduction of GE products such as DNA/GE vaccines and GM feed in commercial fish farming. This may result in a solid basis for the avoidance of potentially undesirable health and environmental effects. If GE can help make aquaculture a sustainable industry, this opens the possibility of positive market and consumer responses. This can best be achieved by involving the stakeholders from the conceptual stage to the commercial stage by facilitating a transparent process whose purpose is to inform research, to identify decision stakes, and to influence design, adoption and implementation of pro-active policy. D 2005 Elsevier B.V. All rights reserved. Keywords: GM feed; DNA vaccines; Scientific uncertainty; Extended peer-review; Ethics; Risk governance; Sustainable development

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . Benefits and risk with the use of DNA vaccines 2.1. Efficacy of DNA vaccines . . . . . . . . 2.2. Induction of specific immune responses .

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T Corresponding author. Fax: +47 77 64 61 00. E-mail address: [email protected] (A.I. Myhr). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.12.032

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2.3. Tolerance . . . . . . . . . . . . . . . . . . . . . . . 2.4. Other immune responses following DNA vaccination. 2.5. Tissue distribution and degradation of plasmid DNA . 2.6. Environmental release of DNA vaccines . . . . . . . 2.7. Lack of transparency and regulatory difficulties. . . . 3. Benefits and risks with distribution of plant-derived feed . . 3.1. Consumption of plant-derived feed by fish . . . . . . 3.2. Unintended changes in the GM plant . . . . . . . . . 3.3. Uptake of transgene(s) by intestinal cells . . . . . . . 3.4. Horizontal gene transfer to bacteria . . . . . . . . . . 4. Risk governance . . . . . . . . . . . . . . . . . . . . . . . 4.1. Public acceptance of GE in aquaculture . . . . . . . . 4.2. Engagement of stakeholders . . . . . . . . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction There is a growing demand for fish and marine products for human and animal consumption. This demand has led to rapid growth of aquaculture, which sometimes has been accompanied by ecological impacts and economic loss due to fish escape and diseases. At present, high quality feed sources have become limiting factors for the further expansion of aquaculture (Naylor et al., 2000). Genetic engineering (GE) strategies, e.g. transgenic fishes, genetically modified (GM) plants as edible vaccines or GM feed, and DNA vaccines may offer a technological solution for some of these problems. Most transgenic fish development has been related to growth promotion in commercially viable fishes (FAO, 2003; Melamed et al., 2002), a trait that is economically interesting since it may reduce production cost per fish and increase feed conversion efficiency. GM plants with improved nutritional qualities, such as increased vitamin content, changed oil profile, and expression of growth-inducing compounds are under development (Fisheries and Oceans Canada, 2003; Ye et al., 2000). In addition, edible vaccines, DNA vaccines, GE vaccines and vaccines based on recombinant proteins hold prospects for immunisation of fish against several diseases (Hew and Fletcher, 2002; Lorenzen et al., 1999a,b). Hence, GE likely will be introduced into commercial aquaculture within a few years.

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At present, there is very limited understanding of the potential ecological effects caused by distribution of DNA vaccines as well as GM feed. For instance, GM feed could affect animal welfare, and gene transfer may occur from DNA in feed or vaccines (NRC, 2002, 2004; Expert Panel of the Royal Society of Canada, 2001). In this paper, we summarise the main benefits and hazards associated with introduction of GE in aquaculture, and identify areas where more research is needed. Our focus is on the scientific and regulatory aspects of risk assessment. We suggest that employment of sustainability as a criterion may influence the scope and frame of risk assessment by providing a normative standard for which harm can be assessed in ecological, socio-economic and ethical terms. The Brundtland Commission defined the concept of sustainable development in the following way; bSustainable development is development that meets the needs of the present without compromising the ability of future generations to meet their own needsQ (WCED, 1987). The Rio declaration of 1992 clarified that governments have a global responsibility for resolving conflicts over the environment in ways that protect the interest of humanity and nature. Accordingly, the Norwegian Gene Technology Act of 1993 prescribes that contribution to sustainability should be assessed in applications of use and release of genetically modified organisms (GMOs). With regard to aquaculture, recommendations for employ-

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ment of sustainability can be found in the Holmenkollen guidelines for sustainable aquaculture (1998) and in the EU (2002) communication, bA strategy for the sustainable development of European aquacultureQ. The EU commission strategy aims at ensuring an environmentally sound industry; inevitably, this includes a prospect for safety requirements for health and the environment, a long-term perspective, and a presumption of democratic decision making. This implies the need to involve stakeholders, i.e., biotechnology firms and research institutions, fish breeders and farmers, governmental offices, and non-governmental organisations (NGOs) such as consumer and environmental groups, from the concept to the commercial stage with the purpose of identifying decision stakes and influencing a proactive policy.

2. Benefits and risk with the use of DNA vaccines It has been estimated that 10% of farmed fish die due to infectious diseases (Leong and Fryer, 1993). For a range of viral diseases, there are no efficient vaccines based on neither live, attenuated virus nor vaccines containing recombinant viral antigens. DNA vaccines represent means to protect fish against viral diseases, and hence improve animal welfare and reduce antibiotic usage and spread of disease. DNA vaccines consist of a bacterial plasmid with a strong viral promoter, the gene of interest, and a polyadenylation/transcriptional termination sequence. The plasmid is grown in bacteria, purified, dissolved in a saline solution, and then administered by direct intramuscular injection of naked DNA to activate protein expression in vivo and ultimately to induce the production of antibodies. After injection of DNA, muscle cells usually perform the protein expression. An example of a DNA vaccine is the plasmid encoding a infectious haematopoietic necrosis virus (IHNV) glycoprotein under the transcriptional control of a cytomegalovirus promoter (pCMV), which has been injected in Atlantic salmon with the purpose of achieving resistance to IHNV (Traxler et al., 1999). DNA vaccines have several advantages: low cost, ease of production and quality control, heat stability, an identical production process for different vaccines,

and the possibility of producing multivalent vaccines by use of plasmid cocktails (Hew and Fletcher, 2002; Liu, 1998; Heppell and Davis, 2000). 2.1. Efficacy of DNA vaccines Following early trials on DNA vaccination in mammalian species, several experiments have been conducted in fish with promising results, such as complete protection against viral diseases (Romøren, 2003 and references therein). In addition, crossprotection against heterologous virus due to induction of innate defence mechanisms (e.g., Mx protein) provides protection for some days after vaccination (LaPatra et al., 2000; Lorenzen et al., 2001). The vaccines have proven to be quite efficient; e.g., Corbeil et al. (2000) have shown that only a single dose of nanogram quantities of IHNV DNA is sufficient to elicit a protective immune response. At present, there is lack of understanding of potential side effects from DNA use in vaccination. It has been shown that following DNA vaccination, the expression and synthesis of several proinflammatory substances are modulated in some vertebrates; e.g., increased IL-1h and IFN-g expression may occur (Lawson et al., 1997; Stacey et al., 1996). It has been suggested that the scavenger receptors may bind most of plasmid DNA after injection, as occurs to other polyanions, prior to internalisation. By using competitive ligand binding assays, it was revealed that other receptors also bind DNA. Toll-like receptor 9 (TLR9) has been shown to bind CpG oligodeoxynucleotides, and there are indications that plasmid DNA containing such motifs binds to TLR9 (Bauer et al., 2001; Zhao et al., 2004). By engaging TLR9, intracellular signalling occurs (Kandimalla et al., 2003); this may induce inflammatory processes other than cytotoxic responses. The TLR9 gene is found in fish (Meijer et al., 2003), but it remains to be elucidated whether plasmid DNA binds to fish TLR9. 2.2. Induction of specific immune responses After DNA vaccination, host cells may produce the antigen of interest, and these antigens may be endocytosed by antigen-presenting cells (APC) and presented on MHC class II molecules to T cells, leading to production of antibodies produced by plasma cells.

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Thus, administration of DNA vaccines has proven to be an effective mean for generation of humoral immune responses specific for the encoded antigen(s) (Russell et al., 1998; Lorenzen et al., 1999a; Fernandez-Alonso et al., 2001; Nusbaum et al., 2002; Verri et al., 2003). A combination DNA vaccine, consisting of multiple discrete plasmids encoding several different antigens of a pathogen, may be employed to induce a broader spectrum of antibody responses. This would be effective for vaccination against viruses that undergo antigenic variation, such as infectious pancreas necrosis virus (IPNV) and infectious salmon anaemia virus (ISAV) (Lee et al., 1996; Wang and Nicholson, 1996, Kibenge et al., 2001). Although there may be high vaccine efficacy (increased survival), the potency (amount of specific antibodies generated) may be low compared with traditional vaccines. To obtain increased potency of the DNA vaccines, one may apply higher doses, or boost or co-administer plasmids encoding cytokines or co-stimulatory molecules. However, opposite effects may result. Xiang and Ertl (1995) observed that co-injection of plasmids encoding IFN-a induced inhibition of the antibody response. 2.3. Tolerance Joosten et al. (1997) has reported that repeated antigen stimulation of fish causes immunological tolerance. Whether this also could be the case after DNA vaccination has not been elucidated so far in fish. Dijkstra et al. (2001) reported that antigen production could be detected up to two years after DNA vaccine injection. The physiological effects of the long-lasting expression of recombinant DNA and proteins have not yet been addressed. Whether there had been a chromosomal integration of new genes during these two years was not examined. 2.4. Other immune responses following DNA vaccination It has been hypothesised that the early protection following DNA vaccination is caused by induction of antiviral mechanisms such as expression of Mx and IFN a/h (LaPatra et al., 2000; Lorenzen et al., 2001). DNA vaccination with a plasmid encoding a viral glycoprotein induced expression of Mx protein in

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kidney and liver (Kim et al., 2000) and in muscle (Boudinot et al., 1998). Apart from the induction of Mx and IFN synthesis and the cytokines important for the polarisation of T cell response, it is not known whether other immune-related molecules also are synthesised following DNA vaccination. However, in a recent trial, a VHS (viral hemorrhagic septicemia) DNA vaccine encoding the viral G protein was injected in Japanese flounder. Of many genes that were up-regulated following injection of the DNA vaccine, especially the genes for NK (natural killer cell), Kupffer cell receptor, macrophage inflammatory factor 1-alpha, CD20 receptor, CD8 alpha chain and CD40 were shown to be induced at levels higher than for control plasmid injection 1–3 d post-injection (Byon et al., 2005). Focus was, unfortunately, not put on examination of up-regulation of genes by the empty plasmid DNA compared to PBS-injected fish. This study used microarray technology to monitor gene expression, and this technology may be beneficial to assess global gene expression. Such microarray technology also is being developed for salmon, carp, rainbow trout, pufferfish and zebrafish, and these microarrays contain a vast number of genes. Application of microarray technology holds promise for monitoring impacts of DNA vaccination on fish, both on short- and long-term time-scales. 2.5. Tissue distribution and degradation of plasmid DNA Several studies have shown that plasmid DNA is rapidly degraded after intramuscular injection in mice (Dupuis et al., 2000). Radiolabelled plasmid DNA was shown to progressively leave the muscle (injection site) and was degraded as soon as 5 min after injection. The major part of the radioactivity was detected in interfibrillar spaces of a large proportion of the muscle at the injection site. While the major part of the injected DNA was rapidly degraded, a minor part bescapedQ to another compartment where it was protected from endogenous DNAses. Besides muscle cells, cells suggested to be antigen-presenting cells (APC) residing between the muscle cells also were shown to contain plasmid DNA in degradative compartments (Dupuis et al., 2000). Later, DNA also was found in draining deep and superficial lymph nodes in rear limbs 3 h after injection. Cells that were

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isolated from these nodes were similar to APC. Even though these cells contained plasmid DNA, only the muscle cells expressed the transgene. Plasmid DNA also was found in liver endothelial cells of mice after intravenous injection (Kobayashi et al., 2001). However, no significant gene expression was observed. This indicates that even though cells contain DNA constructs, the transgenes may not be expressed. The tissue distribution of foreign and endogenous DNA is highly dependent on the localization of receptors and cells active in fluid pinocytosis and endocytosis (Dalmo et al., 1997). The route of DNA administration influences the tissue distribution of the plasmid DNA in fish. After intramuscular injection, fluorescence-labelled plasmid DNA constructs were found mainly in cells located between myofibrils at the site of injection. Minor amounts were found in other tissues such as kidney, gills and heart (R.A. Dalmo, University of Tromsø, unpublished). The peritoneal membrane, hematopoietic organs and connective tissues covering internal organs were highly stained for plasmid DNA following intraperitoneal injection (R.A. Dalmo, University of Tromsø, unpublished). By using a luciferase expression system, it was found that rainbow trout spleen and liver expressed the reporter gene after intramuscular and intraperitoneal injections (Romøren et al., 2003). Unfortunately, the cells expressing luciferase were not characterised in the study, but the authors speculated that mononuclear cells were expressing the reporter. By the introduction of vehicles (liposomes, cationic polymers) that bind DNA, APC such as monocytederived immature dendritic cells may ingest these DNA-coated vehicles and express the transgenes (Strobel et al., 2000), and some of these particles may contain membrane-disrupting chemicals that ease the cytoplasmic delivery of plasmid DNA from endosomes and lysosomes. This, in turn, may lead to increased antigen production by such cells. 2.6. Environmental release of DNA vaccines In aquatic environments, DNA may be distributed over vast areas, distances, and phyla as a result of the relative lack of physical and physiological barriers. In addition, DNA is much more resistant to breakdown in ecosystems than was realised until recently. This is

also the case for aquatic ecosystems (Tappeser et al., 2002; Heinemann and Roughan, 2000). There is a need to initiate experiments with the purpose of investigating the stability of DNA in aquatic systems, horizontal gene transfer (HGT), and uptake of DNA constructs in aquatic organisms, including marine fishes and mammals. 2.7. Lack of transparency and regulatory difficulties In 2003, the U.S. Center for Disease Control (CDC) and Prevention expedited delivery of an experimental veterinary DNA vaccine developed by the CDC and manufactured by Aldevron (Fargo, ND) (Bouchie, 2003). The target for vaccination was the California condor and the purpose was to protect this endangered species from becoming infected with the West Nile virus. In Canada, an IHNV DNA vaccine developed by Novartis is under evaluation for commercial use in aquaculture (M. Horne, Novartis, personal communication). However, the data supporting these applications and the risk assessments that have been done are not publicly available. This lack of transparency also is reflected in the EU, where GM medicinal products lack the public openness central to other GMO applications. The reason for lack of openness is to protect the proprietary interests of the party that has developed the medical product. However, the lack of openness and availability of assessment information may reduce public credibility and confidence in future decisions regarding DNA vaccines. On the contrary, transparency may help to ensure a sound decision by letting stakeholders’ review the data. It will ensure the integrity of the process by disclosing the authorities’ evaluation, and provide a mechanism to improve the knowledge base on which to make the policy decision (Pew Initiative on Food and Biotechnology, 2003). In the U.S. and in Europe, there are directives regulating GMOs, and the discussion about how to regulate DNA vaccines is now at a starting point (British Agriculture and Environment Biotechnology Commission, 2002; Foss and Rogne, 2003). Central to this discussion are the regulatory definitions of bmedicinal productsQ and bgenetic modificationQ and which regulatory system that should be involved. In the U.S., the U.S. Food and Drug Administration has asserted that genetic constructs distributed to animals

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fall under the legal definition of a drug substance. This corresponds with regulations in Europe; the European Agency of Medicinal Products authorise pharmaceuticals based on GE through a centralised procedure. However, as a part of the European procedure, national GMO authorities are involved in evaluating the environmental risks of the medicinal products and the animals receiving it. For instance, the Norwegian Directorate for Nature Management has stated that a DNA-vaccinated animal is to be considered as genetically modified as long as the added DNA is present in the animal (Foss and Rogne, 2003). This may have implications regarding the need for labelling and traceability. In the EU, the regulation on GM food and feed (2002/0173(COD)) specifies that products of animals treated with GM medicinal products need not be labelled as GM food. However, it is not clear whether this labelling exemption is limited to the medicinal products authorised as GMOs, or if it includes DNA-vaccinated products. Accordingly, the current limited scientific understanding of the fate of DNA vaccines has policy implications, and illustrates the need for research with focus on the stability of the DNA construct, on any immunological impacts, and on whether the DNA construct may become integrated into the chromosomes of the recipient organism.

3. Benefits and risks with distribution of plant-derived feed The availability of wild fish species used as feed in fish farming is a limiting factor for future aquaculture expansion (Naylor et al., 2000). The prospect of replacement of marine fish meal with feed stuffs based on plants poses reduction of costs in aquaculture and represents a sustainable alternative source for fish feed. It has been argued that the present practice, which is based on the use of marine fish meal to feed fish instead of being processed into human food, is not in accordance with sustainability (Hannesson, 2003). At present in Norwegian aquaculture, fish feed is mostly based on two different fish species, blue whiting (Micromesistus poutassou) from Norway and anchovy (Engraulis ringens) from Chile and Peru (Tuominen and Esmark, 2003). Although these fish species are not fit for human consumption directly, they are a part of

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food webs important for producing other commercial fish species. That is, removing forage fish from an ecosystem can result in impacts on higher levels in the food web, for instance, in reduction of carnivorous fishes important as human food (Naylor et al., 2000). In addition, anchovy is not competitive on an economic basis in Chile as a local human food resource due to its export to Norway and other nations as fish feed. In Chile, this affects fish availability for human consumption (Tuominen and Esmark, 2003). Accordingly, should the introduction of GE plant-derived feed in Norwegian aquaculture improve the sustainable use of marine resources, it would increase the value of commercial fisheries (by increase of higher tropic level fishes), and enhance the sustainability of livelihoods dependent upon fishing in Chile. 3.1. Consumption of plant-derived feed by fish Alternative feed ingredients from vegetable sources are gaining increasing attention in aquaculture involving carnivorous fish species. However, there are problems related to vegetable sources used in fish feed. Most plants contains anti-nutritional factors, factors that interfere with feed utilisation and have adverse effects of fish welfare (Makkar, 1993). Feed containing soy or canola causes digestive difficulties and reduced digestion of fat in salmonids (Fisheries and Oceans Canada, 2003; Jystad, 2001). Another problem with the use of vegetable sources in fish feed is that it has become increasingly difficult to obtain soy, maize, canola or rapeseed from the global market that are guaranteed free of GM materials. This raises new questions; might there be unintended changes introduced by the genetic modification process in the GM plant, and can the transgene(s) be taken up by intestinal cells or be subject to horizontal gene transfer (HGT) (i.e. nonsexual uptake) to bacteria in the fish intestine? 3.2. Unintended changes in the GM plant By genetic modification, new properties are introduced in the receiving plant. The expression of a constituent (not previously found) in the GM plant may cause unintended changes. Hence, characterisation of the transgene product(s) needs to involve investigation of degradation under conditions of ingestion, processing and storage, and immunological activity and

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sequence homology with proteins known to cause allergy or pathological effects (Aumaitre et al., 2002; Kuiper and Kleter, 2003). The insertion of transgene(s) encoding new protein(s) or other constituents may cause unintended pleiotropic effects, as changes in levels and activities of inherent enzymes, nutrients and metabolites (Haslberger, 2003; Kuiper et al., 2002; the Expert Panel of the Royal Society of Canada, 2001). For instance, transgenic rice containing the soybean glycinin gene exhibited a 20% increase in protein content due to elevated glycine and a 50% increase in vitamin B6 (Momma et al., 1999). The genetic insertion may also be subject to instability of expression, such as silencing of the inserted transgene (Qin et al., 2003). Hence, characterisation of the GM plants needs to involve investigation of: the integrity of the transgene(s), the site of genomic integration and any pleiotropic effects on the host (Kuiper et al., 2002; the Expert Panel of the Royal Society of Canada, 2001). Such characterisation is required by regulatory authorities of most nations before a GM plant is commercialized. Accordingly, it has been suggested that potential ecological impacts by administration of GM feed need to be investigated at five relevant levels. These levels are genome, transcript, protein, metabolite, health impact and environmental impact (Aumaitre et al., 2002; Kuiper et al., 2002; Kuiper and Kleter, 2003) (see Table 1). In addition, feeding experiments need to be an integrated part of a GM food/feed safety assessment. However, only 10 scientific (peer-reviewed) feeding studies on animals have been published; 4 of these articles has been published by the group around Pusztai and Ewen (Domingo, 2000; Pryme, 2003). Only one feeding study involving fish has been published (Sanden et al., 2004). In this study, Atlantic salmon were fed with feed containing 17.2% GM soybean for 6 weeks, and the purpose was to investigate the fate of GM soy DNA fragments. The authors could only detect small DNA fragments (120 and 195 bp) by using PCR methodology on the content of stomach, mid-intestine and distal intestine. No GM soy DNA fragments were detected in liver, muscle or brain tissue. This study needs to be followed up, with feeding over longer periods, investigation of more tissues, such as blood, spleen and gills, using not only PCR technology but also methodology such as Southern blotting.

Table 1 Safety issues regarding GM feed (modified after Kuiper et al., 2002) Issue

Study methods

Characteristics of the genetic modification Change in expression of/by genetic modification, effect on toxicological properties, levels of nutrients and allergens Potential alterations in expression levels Formation of new metabolites

DNA analysis (Southern blotting, sequencing) mRNA and protein analysis (Western blotting, proteomics), amino acid sequence comparisons to toxins and allergens, animal feeding studies Compositional analysis, metabolomics Residue analysis, metabolomics, bioassays Screening for insertion of recombinant DNA, and study of gene transfer in microcosms

Integration and gene transfer between GM feed and bacteria in intestines

3.3. Uptake of transgene(s) by intestinal cells Consumption of GM feed may have unknown impact on the digestive microbial system, and foreign DNA may be taken up by intestinal cells, transferred via the circulatory system, and subsequently distributed to other tissues and organs (NRC, 2002). It has been reported that gene fragments from non-GM plants (N200 bp) have been detected in liver, muscle, blood, kidney and spleen of pigs (Reuter and Aulrich, 2003) and in liver, muscle, kidney, and spleen of chicken and cattle (Einspanier et al., 2001). In human ileostomists (people with a colostomy bag), small fragments of GM soya DNA (180 bp) have been found in the small intestine, and low frequency gene transfer to the gut microflora of gene fragments was detected (Netherwood et al., 2004). However, there was no survival of the GM DNA soya in the large intestine of subjects who had not undergone such surgery. In the gastrointestinal tract of higher vertebrates, specialised cells in the intestinal mucosa (Mcells) may take up low levels of DNA (Nicoletti, 2000), which may endocytose and transport antigens to professional antigen-presenting cells. It remains to be elucidated if there is a similar mechanism of endocytosis in the intestines of fish. 3.4. Horizontal gene transfer to bacteria With regard to fish fed GM feed or to which DNA constructs are distributed as DNA vaccines or by gene therapy, the potential for HGT to bacterial commun-

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ities has to the best of our knowledge not been studied. A large number of different bacteria colonize the intestines of fish. It is possible that these bacteria could be exposed to the recombinant DNA constructs in the digestive system of fish. When monitoring of field trials has included monitoring of horizontal gene transfer from GM plants to soil micro-organisms, the frequency often has been considered to have low impact or to be insignificant. This is because it is easy to demonstrate bno effectQ in highly complex and variable conditions, especially when investigating low frequency or low probability events. Hence, in two recent papers (Heinemann and Traavik, 2004; Nielsen and Townsend, 2004), it is argued that current techniques for sampling and monitoring of HGT from GM plants to soil microorganisms is too insensitive and that more rigorous monitoring may be the only realistic way to detect HGT. Further, they highlight that the frequency of HGT is probably marginally important compared with the selective forces acting on the host receiving the new DNA. This has been illustrated, for instance, with bacterial acquisition of virulence traits and antimicrobial drug resistance (Ochman et al., 2000). Hence, there is a need to initiate studies with focus on the stability of the DNA construct in the fish, and to develop techniques for sampling and monitoring for potential HGT from GM feed to bacteria in the fish intestines.

4. Risk governance Risk governance concerns risk identification, assessment, management and communication. Risk assessment can be further divided into hazard identification, risk characterisation and risk estimation (Covello and Merkhofer, 1993). Risk assessment has been considered a strictly bscientificQ process, while risk management and communication imply value judgement with regard to acceptability, trade-off criteria and adoption of strategies for coping with uncertainty. However, risk assessments are influenced by scientific, ethical, economic, social and political information (Mayo and Hollander, 1991; Wynne, 1992). For instance, risk assessment includes value judgements with regard to the consequences that should be avoided and the process to be applied for risk identification and characterisation.

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With regard to transgenic fish, several publications have been written about the environmental hazards involved and suggestions of how to address the associated risks (ABRAC, 1995; Hallerman et al., 1999; Hallerman and Kapuscinski, 1995; Hedrick, 2001; Kapuscinski et al., 1999; Muir, 2004; Muir and Howard, 1999, 2002; NRC, 2002; Pew Initiative on Food and Biotechnology, 2003). These predictions are based on biological or mathematical models, and one needs to be aware of that there will always be uncertainties related to extrapolations from these predictions and studies to real conditions. With regard to possible ecological consequences of DNA vaccines and GM feed, the issue becomes more complex, since the assignment of probability values to the benefits and adverse effects is difficult or impossible because of the lack of biological and ecological understanding and the complexity of the systems involved. In this context, it is important to be aware that when uncertainties increase, more fundamental kinds of uncertainty, ignorance and indeterminacy, begin to appear (Wynne, 1992; Stirling, 1999): ! Ignorance represents situations where the type of hazard to measure is unknown, i.e., completely unexpected, and unprecedented hazards may emerge. This has historically been experienced with, for example bovine spongiform encephalopathy (BSE), dioxins, and pesticides. These examples underscore the need for research on potential direct and indirect effects on organisms in the aquatic environment by distribution of DNA vaccines and GM feed. ! Indeterminacy, or bgreat uncertaintyQ, describes the inevitable gap between limited experimental conditions and reality, where the consequences of an activity can never be fully predicted. The functional relationships are complex; they depend on inherent biological diversity and may be influenced by climatic and environmental conditions. Therefore, it is crucial that methods for detection and monitoring of impacts by introduction of GE in aquaculture are initiated with the purpose to follow up the performed risk assessment, to map actual ecological effects and to identify unforeseen adverse effects. Monitoring using a system which involves adaptive management, i.e. repeated cycles of goal setting, program design, implementation and evaluation—

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would enable a learning process and improve risk assessment (Kapuscinski et al., 1999). One issue raised by recognizing lack of understanding is the question; whose knowledge is to be recognized and implemented in policy processes? This is a policy decision that government scientific reviewers and regulators will have to address, and they also have to determine the scientific approaches that will be deemed adequate to address them. However, the quantity and quality of the scientific understanding required addressing risk and uncertainty, and the methods and models to be applied, are related to the safety framework the regulatory agencies consider appropriate. For instance, implementation of criteria such as sustainability involves safety requirements, based on the employment of the Precautionary Principle, for health and the environment in a longterm perspective (Myhr and Traavik, 2003a,b). This makes it necessary to involve of a wide base of scientific disciplines (Funtowicz and Ravetz, 1990), such as molecular biologists, marine biologists, immunologists, pathologists, ecologists, philosophers etc., to address uncertain and complex issues. The various methods and models representing the different disciplines may be seen as compatible providers of information and models for studying the problem or the system. With more diversity in the approach, more data will be generated and more responses will be available to understand changing conditions. Such an approach will bring redundancies into the process, involves a challenge to the different disciplines involved to understand and be respectful to other interpretations and approaches, and raises cost of research. However, an integrative approach will improve the knowledge base, and cause development and consensus building about frameworks for evaluating risk and uncertainty regarding GE in aquaculture. 4.1. Public acceptance of GE in aquaculture There are obvious economic advantages arising from application of GE in aquaculture; however, the distribution of DNA vaccines and GM feed also involves potential for ecological adverse effects and ethical dilemmas to society. In general, the introduction of GE has caused significant scientific and public debates. GE associated with medical applications is

more accepted than GM plants, whereas transgenic animals receive most opposition (Anonymous, 2003). The public’s conception of risks related to GE are not limited to technological and economic risks, but also concern factors such as social, cultural and religious values (NRC, 2002; Power, 2003; Sagar et al., 2000; Wynne, 2001). Hence, there may be several issues regarding how consumers will respond to GE in aquaculture that pull in different directions. For instance, the responses to the publication by Hiter et al. (2004) illustrates that the consumer demands safe and reliable food of good quality that has been produced in a sustainable way. Hiter et al. (2004) published a worldwide survey of salmon bought in supermarkets in March 2002. The authors claimed that fish raised in Britain and other northern European countries were contaminated with carcinogenic chemicals that consumers would be unwise to eat more than six times a year. This publication had the immediate effect of reducing salmon sales and still threatens the salmon farming industry. In addition, the growing exploitation of the aquatic environment, it impending conflicts between various users of marine resources, and the importance of coastal areas in fulfilling human needs may also affect public acceptance of GE in aquaculture. Animal welfare and the naturalness of using GE are other factors important for the public. For instance, the notion that animals, apart from having utility or instrumental value, also have intrinsic values and thereby rights at ethical and legal levels is gaining ground. In a GE context, the violation of an animal’s integrity may take place at different levels: the level of the individual animal (phenotypic integrity), the level of the species-specific nature (genotypic integrity) and the level of animality (self-maintenance, self-organisation, independence, animal-type integrity) (Verhoog, 2001). Reference to inherent values, such as species integrity and natural values, has been considered as emotional and subjective, or they have been attributed to Luddism and anti-scientific attitudes (Davies, 2001; Nuffield Council on Bioethics, 1999; Tave, 1993). However, public feelings may be markers of moral values and raise important questions related to bwhat we are allowed to doQ. In this context, the issue of whether GE use may be considered as an extension of traditional breeding and medication, and hence natural, or represents discontinuity is important.

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The bnaturalQ argument also can be understood as a way of expressing fears about harmful consequences, where it is considered that one should be careful about changing nature since bnature knows bestQ. Although public policy in most countries does not explicitly acknowledge ethical issues (Cayford, 2004; Power, 2003; NRC, 2002), these are aspects that might affect acceptance of the commercial introduction of GE in aquaculture and hence are concerns that must be addressed. 4.2. Engagement of stakeholders We suggest that an extension of the scientific peer community might be capable of catalysing debate in meaningful ways between the affected parties, thereby integrating different viewpoints and enabling wider consideration of risk. The bmodelQ for any participatory decision making can be established as described by Rowe and Frewer (2000): the overall aims and objectives (i.e., consultancy, actual decision making, input to an ongoing process, satisfaction of free informed consent); the participants (i.e., professionals, specialists, lay persons, ethical committees, stakeholders) and their representative status (i.e., themselves, organisations, unions, future generations or animals); and the procedure (i.e., closed or by invitation only, open, media access, accountability). For instance, Kapuscinski et al. (2003) have initiated a bSafety First InitiativeQ, a crossindustry and -society partnership with the purpose of developing socially robust, pro-active safety standards that anticipate and resolve safety issues upstream of commercial use of GE. Hence, a framework that can be applied to GE in aquaculture; it includes stakeholders, it identifies bdecision stakesQ, and its purpose is to influence policy that implements safety standards, as for instance sustainability in the consideration of benefits versus risk and uncertainty.

5. Conclusion The most prominent challenge to implementing GE in a sustainability context is how to ensure environmental protection and at the same time achieve economic benefits. This challenge involves active investigation of the proposed benefits and the

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potential adverse effects in a research and policy agenda that encourages broad and long-term thinking. Sustainability also influences socio-economic and ethical issues; these aspects highlight the need to involve stakeholders in risk governance of GE in aquaculture. Such extended peer-review may help to identify; a) at the concept stage, what sorts of GE application may be acceptable, b) during the development stage, what sort of risk-associated research needs to be initiated, and c) at the commercialisation stage, socio-economic, ethical and policy implications that are important to the stakeholders.

Acknowledgement This work is funded by the Norwegian Research Council (Project No. 157157/150).

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