**3. Current management and biological challenges within aquaculture**

Aquaculture industry is currently facing many challenges. These involve animal health and welfare, environmental effects and social effects including economics, global and fair utilization and sharing of resources, rural viability etc. Within the format of a book chapter, only a limited number of challenges can be handled properly. The focus of this section will therefore be limited to biological challenges with emphasis on animal health and welfare, and the management of fish breeding including biotechnological methods.

#### **3.1 Search for improved animal health**

The growth in aquaculture has been accompanied with an increase in diseases caused by pathogens that includes a wide range of bacterial, viral, parasitic and fungal infections. At present diseases in aquaculture are causing big economic problems and are affecting animal welfare significantly. The high density of fish together with the effective pathogen transportation in water creates favourable living conditions for these pathogens. Hence, diseases tend to multiply in farm environments, a situation that represent potential ecological threats both to the farmed fish in itself and to the farm environment including wild fish. In salmon aquaculture disease prevention with antibiotics and chemicals was for

economic development (Kamara et al., 2006). For example, the rapid spread of aquaculture has raised concern about land-use change in coastal areas, impacts on wild fish by escapees, environmental pollution, and extensive use of marine resources for fish feed production. A conceptual framework for sustainable aquaculture has been presented from three perspectives: environmental, economic and sociological (Caffey et al., 1998). This implies that introduction of modern biotechnology must be explored both with regard to the adequacy of present approaches and with regard to the problem solving nature of the new technology. Moreover that there needs to be an awareness that application of modern biotechnology in aquaculture also influences socio-economic values as employment, income, and local economic activity as well as ethics, which are all important elements of sustainability as understood by most users. Hence, sustainable development requires a renewed focus on stakeholders and their needs, it demands clearer understanding of stakeholders perspectives and public concerns as well as attention to issues of institutional structure and representation in decision-making

The next section deals with biological/ecological challenges in aquaculture while section four provides a picture of recent technological developments that may have a bearing on these challenges. In section five we present international and domestic regulations relevant to both modern biotechnology and the access issues, thus pertaining to the discussion of sustainability. We then briefly account the present structural developments and management trends within aquaculture. With this broad framework in mind, we turn to examine actor perceptions of how biotechnology and IPR may affect sustainability in aquaculture. This section builds on surveys and interviews with key actors. Then in section eight we highlight some of the major issues for understanding how IPR and biotechnology may affect sustainability in aquaculture. Finally we discuss implications and give some recommendations for how developments in biotechnology and IPR in aquaculture can

**3. Current management and biological challenges within aquaculture** 

and the management of fish breeding including biotechnological methods.

Aquaculture industry is currently facing many challenges. These involve animal health and welfare, environmental effects and social effects including economics, global and fair utilization and sharing of resources, rural viability etc. Within the format of a book chapter, only a limited number of challenges can be handled properly. The focus of this section will therefore be limited to biological challenges with emphasis on animal health and welfare,

The growth in aquaculture has been accompanied with an increase in diseases caused by pathogens that includes a wide range of bacterial, viral, parasitic and fungal infections. At present diseases in aquaculture are causing big economic problems and are affecting animal welfare significantly. The high density of fish together with the effective pathogen transportation in water creates favourable living conditions for these pathogens. Hence, diseases tend to multiply in farm environments, a situation that represent potential ecological threats both to the farmed fish in itself and to the farm environment including wild fish. In salmon aquaculture disease prevention with antibiotics and chemicals was for

processes.

contribute to sustainability.

**3.1 Search for improved animal health** 

many years the solution preferred. However, the potential pollution associated with chemicals and the excessive use of antibiotics together with the emergence of multiple resistance to antibiotics created concerns and initiated a search for alternative ways, as selection for increased disease resistance, to deal with the problem.

Selection for increased disease resistance in fish has mainly been based on challenge tests carried out under controlled conditions. Challenge-tested fish cannot be used as parents for the next generation of elite salmon, meaning that selection cannot be applied directly on the breeding candidates. To circumvent this problem, geneticists have been searching for genes controlling the degree of resistance to different diseases. Markers for such genes may be ideal criteria for selection, because they can be applied directly without requiring challenge testing (see also section 4.2). Thus, the accuracy of selection can be increased while the need to sacrifice fish in challenge tests is reduced.

Selection for genetic disease resistance has been emphasized in Norwegian salmon breeding since 1995. In 2007, Moen et al. (2009) identified markers for a gene that explains most (80 %) of the genetic variation in resistance to infectious pancreatic necrosis (IPN) in both fry and post-smolts. Based on these findings, Aqua Gen has developed and applied a tool using these markers for selecting IPN-resistant fish directly. This tool can, with very high accuracy, determine whether individual fish have zero, one or two copies of the gene variant (allele) that give high resistance. This approach may also be useful for pancreas disease (PD), which is an important economic disease of farmed Atlantic salmon that cause significant losses through mortality and reduced production (SalmoBreed, 2011).

Currently, salmon lice (*Lepeophtheirus salmonis*) represent a major health and welfare problem in the salmon industry. Furthermore, it is also an ecological problem, since the lice multiply in fish farms, and then spread to the wild salmon population. Chemical treatment is commonly used to combat the lice, but use of biological measures such as cleaner fish has increased lately due to development of resistant lice to the chemicals. However, moderate genetic variation has been shown for resistance to the salmon louse, and thus it may be possible to reduce problems caused by lice through selective breeding programs (Kolstad et al., 2005). Breeding for disease and parasite resistance in Norwegian salmon and trout is considered to be important for the fish themselves, producers and consumers alike and would increase the sustainability of the industry, and the know-how could be transferred to other aquaculture species.

#### **3.2 Genetic diversity and fish breeding strategies**

Substantial long term selection responses of 10-15% higher growth rate per generation have been documented for several species of farmed fish, such as Atlantic salmon in Norway since the 1970'ies and Nile tilapia in Asia since the 1990'ies. Highly favorable benefit cost ratios ranging from 8 to 60 is reported for fish breeding. Due to the high fertility of fish and convenience of handling and distributing seeds, such high benefit cost ratio can be obtained for fish breeding programs. For this it is, however, important to note that an efficient dissemination structure and organization reaching a high number of farmers is crucial. In Norway, seed from the improved farmed salmon was sold to the farmers as eyed eggs and smolts. As the genetic gain became more apparent, the demand for genetically improved salmon increased rapidly during the first decade. Until the market demand and dissemination was appropriately developed, public funding allowed for establishment of the salmon breeding program. During the early 1980'ies, the Norwegian Fish Farmers Association got involved in the program, and in 1992, the breeding program was turned into a private company, Aqua Gen, a sustainable business with long term profitability. The GIFT (Genetic Improvement of Farmed Tilapia) program is another example of a successful breeding program resulting from public funded research and technology development. The GIFT seeds have been disseminated to several countries in Asia and Latin America to support intensive large scale farms and small subsistence farms.

Genetic variation is essential for selection response, and a sufficiently large and genetically diverse breeding population is, therefore, fundamental when establishing and running an animal breeding program. A breeding design with appropriate family structure is critical to maintain a large effective population size and obtain a long-term selection response with low rates of inbreeding in fish breeding. For mass selection, Bentsen & Olesen (2002) concluded from a simulation study that a minimum of 50 families (pairs of parents) are required to prevent inbreeding and obtain a long-term response in a mass selection program for aquaculture. Gjerde et al. (1996) presented optimum designs for fish breeding programs with constrained inbreeding and mass selection. Various breeding designs for betweenfamily, within-family, and combined selection (between- and within-family) are presented and evaluated by Bentsen & Gjerde (1994). Combined family based selection designs may improve the accuracy of selection substantially, particularly for traits with low heritability. Due to the higher probability of selecting large numbers of sibs from a few families the number of broodstock selected per family needs to be restricted to avoid a high rate of inbreeding and reduced genetic variation.

Less than 10% of the fish stocked for aquaculture in 2005 originated from family based selection programs (Gjedrem et al., 2011). This situation has not improved much recently, and for many aquaculture species with huge production quantities, such as carps, only a few efficient selection programs are active. Furthermore, the effective population sizes are often limited and in some cases too low, because the high reproductive capacity allows the use of a low number of broodstock. Such populations may still gain sufficient short-term advantage above non-improved populations and capture much of the market share. In turn, this discourages further genetic introductions into the breeding nucleus. Long-term inbreeding and loss of genetic variability because of genetic drift may then affect performance and further the long-term genetic progress. In such populations, strategies for continuous (re)introduction of genetic variability from outside the breeding nucleus without adverse performance consequences are, therefore, required. Furthermore, initiation of additional breeding programs is expected for different environments for the most important farmed species and this may improve the situation.

#### **3.3 Prospects for genetic improvement of fish welfare**

During the initial stages, breeding programmes for farmed fish usually focus on improving productivity traits such as growth. During later stages, disease resistance, survival, and product quality traits are often emphasized during selection to develop a more robust fish. Domesticated fish fit better for a life in captivity and farm environment, and are therefore less stressed and will be more robust and perform better with respect to

market demand and dissemination was appropriately developed, public funding allowed for establishment of the salmon breeding program. During the early 1980'ies, the Norwegian Fish Farmers Association got involved in the program, and in 1992, the breeding program was turned into a private company, Aqua Gen, a sustainable business with long term profitability. The GIFT (Genetic Improvement of Farmed Tilapia) program is another example of a successful breeding program resulting from public funded research and technology development. The GIFT seeds have been disseminated to several countries in Asia and Latin America to support intensive large scale farms and small

Genetic variation is essential for selection response, and a sufficiently large and genetically diverse breeding population is, therefore, fundamental when establishing and running an animal breeding program. A breeding design with appropriate family structure is critical to maintain a large effective population size and obtain a long-term selection response with low rates of inbreeding in fish breeding. For mass selection, Bentsen & Olesen (2002) concluded from a simulation study that a minimum of 50 families (pairs of parents) are required to prevent inbreeding and obtain a long-term response in a mass selection program for aquaculture. Gjerde et al. (1996) presented optimum designs for fish breeding programs with constrained inbreeding and mass selection. Various breeding designs for betweenfamily, within-family, and combined selection (between- and within-family) are presented and evaluated by Bentsen & Gjerde (1994). Combined family based selection designs may improve the accuracy of selection substantially, particularly for traits with low heritability. Due to the higher probability of selecting large numbers of sibs from a few families the number of broodstock selected per family needs to be restricted to avoid a high rate of

Less than 10% of the fish stocked for aquaculture in 2005 originated from family based selection programs (Gjedrem et al., 2011). This situation has not improved much recently, and for many aquaculture species with huge production quantities, such as carps, only a few efficient selection programs are active. Furthermore, the effective population sizes are often limited and in some cases too low, because the high reproductive capacity allows the use of a low number of broodstock. Such populations may still gain sufficient short-term advantage above non-improved populations and capture much of the market share. In turn, this discourages further genetic introductions into the breeding nucleus. Long-term inbreeding and loss of genetic variability because of genetic drift may then affect performance and further the long-term genetic progress. In such populations, strategies for continuous (re)introduction of genetic variability from outside the breeding nucleus without adverse performance consequences are, therefore, required. Furthermore, initiation of additional breeding programs is expected for different environments for the most important

During the initial stages, breeding programmes for farmed fish usually focus on improving productivity traits such as growth. During later stages, disease resistance, survival, and product quality traits are often emphasized during selection to develop a more robust fish. Domesticated fish fit better for a life in captivity and farm environment, and are therefore less stressed and will be more robust and perform better with respect to

subsistence farms.

inbreeding and reduced genetic variation.

farmed species and this may improve the situation.

**3.3 Prospects for genetic improvement of fish welfare** 

growth and survival. Hence, maintaining good fish welfare by reducing stress load and making sure the fish is thriving will be a key to promote a more robust fish and profitable farming.

Huntingford et al. (2006) list several factors in aquaculture that represent fish welfare challenges including aggressive interactions, handling and removal from water, diseases and permanent adverse physical states and possibly increased levels of aggressiveness due to selection for fast growth. However, it has been shown in both salmon and cod that after a few generations of selection for growth and domestication in hatcheries and farms, we obtain calmer, less aggressive carnivorous fish. In a review of the effects of domestication on aggressive and schooling behaviour in fish, Ruzzante (1994) conclude that domestication may strongly affect behavioural traits, but it is the intensity of the behaviour rather than the behavioural pattern itself that is affected. Olesen et al. (2011) emphasised possible correlated effects on stress coping for fast growing fish.

Selection for high production efficiency in terrestrial animals is known to give undesirable effects in traits like health and reproduction (Rauw et al., 1998). However, in the Nordic countries broader breeding goals including functional and welfare traits have been selected for. Olesen et al. (2000) discussed definition of breeding goals for sustainable farm animal production, and suggest a procedure including non-market values for appropriate weighing of traits providing public goods (e.g. welfare traits). Since 1995, farmed salmon in Norway have been selected for resistance to diseases. Such selection will obviously reduce stress and suffering connected to diseases. Particularly for farmed fish, there is a lack of information on genetic variances and covariances of many welfare related traits such as behaviour (e.g. aggression) and stress coping. Consequently, we do not know possible unfavourably correlated responses in some fish welfare indicators, e.g. poorer ability to cope with stress, resulting from the current selection for productivity traits. Hence, more knowledge and research is needed on fish welfare traits and their genetic parameters. Regarding survival and maturation, behaviour, dominance, aggressiveness and activity level, it is reported genetic differences between wild and farmed salmon (Fleming & Gross, 1992; McGinnity et al., 1997; Metcalfe et al., 2003; Petersson & Järvi, 2006). Hatchery reared salmonids showed a weaker antipredator response (Johnsson et al., 1996) and less physiological stress due to higher stocking densities (Mazur & Iwama, 1993) when compared to the wild. As farmed fish adapt to the farm environment, such domesticated fish will suffer less in the farm environment. Relevant fish welfare indicators or traits that currently can be taken into account in selective breeding are growth, survival (or mortality), social interactions/behaviour (e.g. cannibalism for carnivorous species) and frequency of injuries (e.g. fin injuries) (Turnbull et al., 1998).

#### **3.4 Animal welfare and animal ethics**

Promoting good husbandry practices and ensuring the welfare of farmed fish are wellestablished parts of the European Union policy for sustainable aquaculture development. However, there are often conflicts and trade-offs between short term profit of the industry and demand for cheap animal products on one hand and animal welfare on the other, that the animals do not gain from. Animal welfare has mostly been discussed in relation to research animals, land based animals for food production, and pets. Some of these issues will be highlighted before we move into implications of fish farming on animal welfare.

An important question with regard to animal husbandry is if it is morally legitimate to use animals merely as a resource or means to meet our needs, or if there are moral considerations that place restrictions on such an approach. Many difficult questions have arisen with regard to animals' intrinsic value. Assuming that animals do have intrinsic value, all encroachments on their lives (by humans) become moral issues and demand carefully considered answers and actions. The Norwegian Animal Welfare Act of 2010, states that animals have an intrinsic value. This term implies that animal welfare must be prioritised irrespective of the value the animal may have for people, which also contributes to clarifying the animal's status.

The word 'welfare' is derived from *well* + *fare*, i.e., how well (or dignified) an animal 'fares' (travels) through life. How well is an animal able to regulate its biological functions in relation to its environment? A function based definition of animal welfare is given (Broom, 1986): 'The welfare of an animal is its state as regards its attempts to cope with its environment'. Other definitions focus on an animal's subjective experience or awareness of its condition (feeling based) and/or on whether it can live a natural life (nature based). Hence, the term 'animal welfare' applies to both the mental/emotional and physical health of the individual animal or the animal's condition while trying to cope with its environment. The term also includes behaviour, as well as physiological and immunological factors. In this context, health is defined more broadly than merely the absence of disease. An important basis for ensuring animal health is the animals' well-being. It also includes positive welfare, with the implication that denying animals positive experiences and stimuli is also an ethical problem with regard to animal protection. 'Animal protection' is here seen as the protection of the emotional and the physical health of individual animals.

Most current animal ethicists use animal ability of sentience for ascribing direct moral considerations. Lund et al. (2007) claimed that fish welfare should be given serious moral considerations depending on their possession of the morally relevant similarities of sentience. The same authors reason further that fish are likely to be sentient and therefore deserve serious consideration. They also concluded from a simple risk analysis that the probability that the fish can feel pain is not negligible, and that if they really experience pain the consequence is great due to the possibly high number of suffering animals. Hence, farmed fish should be given the benefit of doubt. Even from a more egoistic standpoint, we can argue for fair treatment of animals. If we inflict suffering upon animals, we violate human dignity and may contribute to the development of a crueller society, as also indicated by Mahatma Gandhi ('The greatness of a nation and its moral progress can be judged by the way its animals are treated.').

### **4. Biotechnology in aquaculture and its role in innovation**

Modern biotechnology does involve new tools to meet several of the challenges that aquaculture is at present striving with. In this chapter we will therefore limit our presentation to breeding and vaccine development and then to the promising possibilities by chromosome manipulation, DNA marker selection and genetic engineering.

#### **4.1 Reproduction technology involving chromosome manipulation**

In most aquaculture species, external fertilization is natural, and opens many powerful methods of genetic engineering, including manipulation of chromosome number such as

An important question with regard to animal husbandry is if it is morally legitimate to use animals merely as a resource or means to meet our needs, or if there are moral considerations that place restrictions on such an approach. Many difficult questions have arisen with regard to animals' intrinsic value. Assuming that animals do have intrinsic value, all encroachments on their lives (by humans) become moral issues and demand carefully considered answers and actions. The Norwegian Animal Welfare Act of 2010, states that animals have an intrinsic value. This term implies that animal welfare must be prioritised irrespective of the value the animal may have for people, which also contributes

The word 'welfare' is derived from *well* + *fare*, i.e., how well (or dignified) an animal 'fares' (travels) through life. How well is an animal able to regulate its biological functions in relation to its environment? A function based definition of animal welfare is given (Broom, 1986): 'The welfare of an animal is its state as regards its attempts to cope with its environment'. Other definitions focus on an animal's subjective experience or awareness of its condition (feeling based) and/or on whether it can live a natural life (nature based). Hence, the term 'animal welfare' applies to both the mental/emotional and physical health of the individual animal or the animal's condition while trying to cope with its environment. The term also includes behaviour, as well as physiological and immunological factors. In this context, health is defined more broadly than merely the absence of disease. An important basis for ensuring animal health is the animals' well-being. It also includes positive welfare, with the implication that denying animals positive experiences and stimuli is also an ethical problem with regard to animal protection. 'Animal protection' is here seen

as the protection of the emotional and the physical health of individual animals.

a nation and its moral progress can be judged by the way its animals are treated.').

by chromosome manipulation, DNA marker selection and genetic engineering.

**4.1 Reproduction technology involving chromosome manipulation** 

**4. Biotechnology in aquaculture and its role in innovation** 

Most current animal ethicists use animal ability of sentience for ascribing direct moral considerations. Lund et al. (2007) claimed that fish welfare should be given serious moral considerations depending on their possession of the morally relevant similarities of sentience. The same authors reason further that fish are likely to be sentient and therefore deserve serious consideration. They also concluded from a simple risk analysis that the probability that the fish can feel pain is not negligible, and that if they really experience pain the consequence is great due to the possibly high number of suffering animals. Hence, farmed fish should be given the benefit of doubt. Even from a more egoistic standpoint, we can argue for fair treatment of animals. If we inflict suffering upon animals, we violate human dignity and may contribute to the development of a crueller society, as also indicated by Mahatma Gandhi ('The greatness of

Modern biotechnology does involve new tools to meet several of the challenges that aquaculture is at present striving with. In this chapter we will therefore limit our presentation to breeding and vaccine development and then to the promising possibilities

In most aquaculture species, external fertilization is natural, and opens many powerful methods of genetic engineering, including manipulation of chromosome number such as

to clarifying the animal's status.

haploids and polyploids with three (tetraploids) and four sets of chromosomes (tetraploids). Furthermore, animals with chromosomes from only the dam (gynogenesis) or from only the sire (androgenesis) can be produced. A more comprehensive overview of the techniques involved and applications is given by Refstie & Gjedrem (2005).

Production of sterile fish may solve the problem of escaped fish interacting with wild fish and the need for protecting improved genetic material against 'piracy copying'. Early sexual maturation cause problems in commercial farming due to poorer filet quality, higher mortality and reduced growth. Sterile triploid fish will not produce gonads and can continue growing and being slaughtered at any time. For many species, one of the sexes gets earlier sexual mature and hence lower growth and body weights. Chromosome manipulations can be used to produce either all male (as in e.g. tilapia and salmon) or all female fish (as in e.g. halibut, hake and angler fish) depending on the sex preferred. Triploid trout and all male tilapia are the most common applications of chromosome manipulations in aquaculture.

Questions of cost/benefit analysis that need to be addressed before using reproduction technology are: is one gender more highly priced by the market; is one gender of higher production value to producer; is gender determination known for the species or is it mainly environmentally induced, will 'clean green' market perception/sales pitch be jeopardized by the use of this technology (Robinson, 2002).

### **4.2 Application of molecular genetics in aquaculture breeding**

The two areas of modern biotechnology that has been expected to have most significant impact on genetic improvement of aquaculture species are DNA markers and transgenics (Hayes & Andersen, 2005). A DNA marker is an identifiable physical location on a chromosome whose inheritance can be monitored (Hyperdictionary, 2003). A comprehensive overview of DNA markers and linkage mapping together with a discussion of potential applications of DNA markers in aquaculture breeding programmes is given by Hayes & Andersen (2005). Furthermore, whole genome sequencing and application of genomics in aquaculture breeding programs is discussed by Quinn et al. (2011, see chapter in this book). As mentioned, DNA markers have already been applied in aquaculture breeding for direct and highly accurate selection of IPN-resistant fish (Aqua Gen, 2010). So called marker assisted selection (MAS) can double genetic gain for traits that can not be measured on selection candidates (e.g. disease resistance), because it utilizes the between family variance, and may also contribute to reduce inbreeding. For MAS, quantitative trait loci (QTL) must be mapped and their effect determined. This is not the case for genomic selection (GS), where the effects of a large number of loci are first estimated using a test group. Selection can then be carried out on genome wide breeding values of the breeding candidates predicted as the sum of the marker effects estimated, assuming an additive genetic model (Meuwissen et al., 2001). However, the high genotyping costs for GS has so far limited its application in aquaculture breeding. Therefore, a scheme with pre-election of parents for growth combined with selective genotyping of large and pooled family groups has been suggested to obtain high accuracies while reducing number of genotypes and costs many fold (Sonesson et al., 2010).

Furthermore, application of new tools of molecular genetics for gaining understanding about genetic regulation of complex traits such as disease resistance may be important (see section 3.1).

#### **4.3 Genetically modified organisms**

The possibilities within modern biotechnology related to the ability to identify genes endowing specific phenotypes together with projects intended to map genomes have opened the possibility for the development of genetically modified organisms (GMOs). Of special relevance for aquaculture are research and development of transgenic fish, GM vaccines (here also included DNA vaccines) as well as present and future GM plants to be used in feed. Genetic engineering can also be a useful tool for increased use of IPR, as it may make it easier to fulfil patent criteria such as the inventive step and the demand for reproducibility.

#### **4.3.1 Introduction of transgenic fish**

Most focus on trangenic fish is on the possibilities for enhancement of the quality of cultured stocks by improving growth rate and increasing resistance to disease and stress (Melamed et al., 2002). Improved growth rate has been possible by the introduction of growth hormone (GH) genes, in species such as Atlantic salmon, coho salmon, Nile tilapia and hybrid tilapia. The most known example of transgenic salmon is the *AquAdvantage*, developed by Aqua Bounty, which contains a gene construct composed of the regulatory elements of an ocean pout antifreeze protein gene controlling a chinook salmon GH gene. The antifreeze promotor enables stimulation of the growth hormone gene also during cold periods with the result that the transgenic salmon grows much faster than its non-GM counterpart. The company is seeking approval for commercial use of this transgenic fish, and the application has been under evaluation in more than ten years by the US Food and Drug Administration (Niiler, 2000). Other highly relevant approaches are development of disease and parasite resistant fish. At present there is a lack of understanding of genes responsible for disease resistance in fish and different strategies are discussed. These strategies include antisense technology for production of complementary RNA for foreign RNA, expression of antimicrobial substances and peptides (as lysozyme) and efforts to increase production of the fish cytokines and other genes involved in immune defence. Moreover transgenic approaches that combine interesting characteristics, as enhanced growth and disease resistance, together with approaches for development of sterile fish or fish where reproductive activity can be down-regulated is also highly relevant since this will minimise the risk of transgenic fish breeding with wild populations after accidental release or escape.

The potential of transgenic fish to escape and enter the natural environment is an important concern for regulators (Le Curie-Belfond et al., 2009). Unless transgenic fish is used in contained facilities, transgenic fish will certainly escape into the environment. The environmental impact is difficult to predict and will depend on the number of escaped fish, their phenotypic characteristics (related to ability for reproduction and survival over time), and the aquatic biodiversity present in the receiving ecosystem (Kapuscinski & Brister, 2001).

Another potential problem related to transgenic fish that is disease or parasite resistant may be similar to what has been experienced with insect resistant crops (Le Curie-Belford et al., 2009). It has for example been reported that insects have developed resistance to insect resistant crops. Hence, the benefit achieved may over time develop into a long-term problem. If the same unexpected events develops with disease or parasite resistant fish the consequence will be a need for more or other antibiotics and chemicals to cope with resistant pathogens and parasites or new emerging pathogens.

Development of transgenic fish does also raise ethical concern. One implication by the genetic modification process itself is that it may affect fish welfare, behaviour and reproduction (see also Le Curie-Belford et al., 2009). It has been reported pleiotropic effects as changes in coloration, cranial, opercula and lower jaw deformation in transgenic coho salmon (Devlin et al., 1995). Concerns have also been raised that genetic modification strategies may affect the animal's integrity (Verhoog, 2001). This is controversial, and for example Sandøe & Holtug (1998) argue that only welfare of animals and humans are relevant ethical considerations, and that these considerations imply to:


324 Aquaculture

The possibilities within modern biotechnology related to the ability to identify genes endowing specific phenotypes together with projects intended to map genomes have opened the possibility for the development of genetically modified organisms (GMOs). Of special relevance for aquaculture are research and development of transgenic fish, GM vaccines (here also included DNA vaccines) as well as present and future GM plants to be used in feed. Genetic engineering can also be a useful tool for increased use of IPR, as it may make it easier to fulfil patent criteria such as the inventive step and the demand for

Most focus on trangenic fish is on the possibilities for enhancement of the quality of cultured stocks by improving growth rate and increasing resistance to disease and stress (Melamed et al., 2002). Improved growth rate has been possible by the introduction of growth hormone (GH) genes, in species such as Atlantic salmon, coho salmon, Nile tilapia and hybrid tilapia. The most known example of transgenic salmon is the *AquAdvantage*, developed by Aqua Bounty, which contains a gene construct composed of the regulatory elements of an ocean pout antifreeze protein gene controlling a chinook salmon GH gene. The antifreeze promotor enables stimulation of the growth hormone gene also during cold periods with the result that the transgenic salmon grows much faster than its non-GM counterpart. The company is seeking approval for commercial use of this transgenic fish, and the application has been under evaluation in more than ten years by the US Food and Drug Administration (Niiler, 2000). Other highly relevant approaches are development of disease and parasite resistant fish. At present there is a lack of understanding of genes responsible for disease resistance in fish and different strategies are discussed. These strategies include antisense technology for production of complementary RNA for foreign RNA, expression of antimicrobial substances and peptides (as lysozyme) and efforts to increase production of the fish cytokines and other genes involved in immune defence. Moreover transgenic approaches that combine interesting characteristics, as enhanced growth and disease resistance, together with approaches for development of sterile fish or fish where reproductive activity can be down-regulated is also highly relevant since this will minimise the risk of transgenic fish breeding with wild populations after accidental

The potential of transgenic fish to escape and enter the natural environment is an important concern for regulators (Le Curie-Belfond et al., 2009). Unless transgenic fish is used in contained facilities, transgenic fish will certainly escape into the environment. The environmental impact is difficult to predict and will depend on the number of escaped fish, their phenotypic characteristics (related to ability for reproduction and survival over time), and the aquatic biodiversity present in the receiving ecosystem (Kapuscinski &

Another potential problem related to transgenic fish that is disease or parasite resistant may be similar to what has been experienced with insect resistant crops (Le Curie-Belford et al., 2009). It has for example been reported that insects have developed resistance to insect resistant crops. Hence, the benefit achieved may over time develop into a long-term

**4.3 Genetically modified organisms** 

**4.3.1 Introduction of transgenic fish** 

reproducibility.

release or escape.

Brister, 2001).


When weighing conflicting ethical concerns we often have to compromise between efficiency and animal welfare, where different ethical theories of animal ethics may affect acceptance of biotechnology on animals (Sandøe & Christensen, 2008):


In the Norwegian Animal Welfare Act (2010) §25, the following is stated about animal breeding: 'Reproduction, including through methods of gene technology, shall not be carried out in such a way that it:


This may reflect a hybrid view including utilitarian (first two points) and animal rights based (third point) views. The first point (animals' normal functions) may also have an element of respect for nature. One may therefore argue that such a hybrid may be the base for the public ethical view on using transgenic animals in Norway.

#### **4.3.2 IPR and the introduction of GM and DNA vaccines**

Modern biotechnology provides tools both for rapid detection and identification of disease and holds promises for new and improved vaccines. Generally spoken, there are two strategies for GM vaccine development: The first is represented by gene-deleted bacteria/ viruses to be used for homologous vaccination, i.e. to achieve protective immunity against the GM vaccine itself. The other strategy involves development of recombinant vaccines by genetic engineering where a gene that is immunologically targeted to a) be expressed after insertion in bacteria or yeast and the proteins produced are then further incorporated into a vaccine preparation, b) be inserted in a virus or bacteria by recombination and the recombinant virus or bacteria is then used as a vaccine, and c) DNA vaccine.

In aquaculture the most effective vaccines at present are multivalent (contains several genes of interest) and target salmon, there is also ongoing research to develop vaccines for other species (as seabass, tilapia, grouper etc.). Present approaches are, however, limited to some bacterial and viral diseases while there are no vaccines against parasites of fish. Especially intracellular pathogens, such as virus and some bacteria, have been found to be difficult to eradicate with traditional vaccines. Hence, DNA vaccines may offer a technological solution to these problems. An example of a DNA vaccine is the plasmid encoding infectious haematopoietic necrosis virus (IHNV) glycoprotein under 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). 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). A combination DNA vaccine, consisting of multiple plasmids encoding several different antigens of a pathogen, holds prospect for inducing a broad spectrum of antibody responses, and hence be effective for vaccination against viruses that undergo antigenic variation (e.g infectious pancreas necrosis virus (IPNV) and infectious salmon anaemia virus (ISAV)) (Kibenge et al., 2001).

The development of both GM and DNA vaccines against infectious fish diseases has several attractive benefits: low cost, ease of production and improved quality control, heat stability, identical production processes for different vaccines, and the possibility of producing multivalent vaccines (Hew & Fletcher 2002; Kwang 2000). On the other hand, there is at

In the Norwegian Animal Welfare Act (2010) §25, the following is stated about animal breeding: 'Reproduction, including through methods of gene technology, shall not be

• changes genes in such a way that they influence the animals' physical or mental

This may reflect a hybrid view including utilitarian (first two points) and animal rights based (third point) views. The first point (animals' normal functions) may also have an element of respect for nature. One may therefore argue that such a hybrid may be the base

Modern biotechnology provides tools both for rapid detection and identification of disease and holds promises for new and improved vaccines. Generally spoken, there are two strategies for GM vaccine development: The first is represented by gene-deleted bacteria/ viruses to be used for homologous vaccination, i.e. to achieve protective immunity against the GM vaccine itself. The other strategy involves development of recombinant vaccines by genetic engineering where a gene that is immunologically targeted to a) be expressed after insertion in bacteria or yeast and the proteins produced are then further incorporated into a vaccine preparation, b) be inserted in a virus or bacteria by recombination and the

In aquaculture the most effective vaccines at present are multivalent (contains several genes of interest) and target salmon, there is also ongoing research to develop vaccines for other species (as seabass, tilapia, grouper etc.). Present approaches are, however, limited to some bacterial and viral diseases while there are no vaccines against parasites of fish. Especially intracellular pathogens, such as virus and some bacteria, have been found to be difficult to eradicate with traditional vaccines. Hence, DNA vaccines may offer a technological solution to these problems. An example of a DNA vaccine is the plasmid encoding infectious haematopoietic necrosis virus (IHNV) glycoprotein under 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). 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). A combination DNA vaccine, consisting of multiple plasmids encoding several different antigens of a pathogen, holds prospect for inducing a broad spectrum of antibody responses, and hence be effective for vaccination against viruses that undergo antigenic variation (e.g infectious pancreas necrosis virus (IPNV) and infectious salmon

The development of both GM and DNA vaccines against infectious fish diseases has several attractive benefits: low cost, ease of production and improved quality control, heat stability, identical production processes for different vaccines, and the possibility of producing multivalent vaccines (Hew & Fletcher 2002; Kwang 2000). On the other hand, there is at

functions in a negative way, or passes on such genes, • reduces the animals' ability to practise natural behaviour, or

for the public ethical view on using transgenic animals in Norway.

recombinant virus or bacteria is then used as a vaccine, and c) DNA vaccine.

**4.3.2 IPR and the introduction of GM and DNA vaccines** 

carried out in such a way that it:

• stimulates general ethical reactions.

anaemia virus (ISAV)) (Kibenge et al., 2001).

present a limited scientific understanding of the fate of such vaccines after injection into the animal. There is a need for research with focus on the stability of the vectors and of the DNA construct, and if there are any unintended immunological impacts, the biological effect of the vaccine after injection (e.g. persistence, distribution, expression and integration) (Gillund et al., 2008a, 2008b). For GM vaccines it is important to investigate potential recombination with relatives and spread in the environment by vectors (Myhr & Traavik, 2011).

An example of the socioeconomic dilemmas relating to IPR, is the case of Intervet's patent on the Pancreas Disease (PD) virus and whether it is a potential barrier to further development of a PD vaccine or not. As the patent has been given on the virus itself, this gives Intervet full monopoly on developing vaccines against Pancreas Disease (PD). Pharmaq wanted a license to produce a PD vaccine and asked the competition authorities for a compulsory licence without success. They argued that the current Intervet vaccine is inefficient and their production is insufficient, and that their own vaccine is superior in terms of time, costs and animal welfare as it can be given as a component of one injection of multi-vaccination (Haavind Vislie, 2008). The Ministry of Fisheries and Coastal Affairs (FKD) was not directly involved although the competition authorities asked for their opinion. In a trial case, Pharmaq have emphasised that they apply a variant of the PD virus that is different from PD virus patented by Intervet. The case is still open as this book is published.
