APPENDIX G
RESEARCH AND DEVELOPMENT OF APPROPRIATE GENETIC BIOTECHNOLOGIES FOR THE FISHERY SECTOR IN DEVELOPING COUNTRIES

Introduction

1. Biotechnology in fisheries and aquaculture represents a range of technologies that present opportunities to increase growth rate in farmed species, to improve nutrition of aquafeeds, to improve fish health, to help restore and protect environments, to extend the range of aquatic species and to improve management and conservation of wild stocks. The focus here will be on genetic biotechnologies, with a brief treatment of related reproductive and gene banking technologies, and the appropriateness of their application in developing countries. It is important to note that developing countries produce more fishery products from aquaculture, inland capture fisheries and marine capture fisheries than developed countries. The coverage of the biotechnologies here is not comprehensive, but will provide general information on which the ACFR can discuss.

2. The vast majority of aquatic genetic resources are found in wild populations of fishes, invertebrates and aquatic plants. Fishstat, the FAO database on fishery statistics, lists 1,235 taxa of common aquatic species that are harvested by humans in major fisheries; thousands more species are taken by small-scale fishers. It also contains information on 440 species that are farmed, but just 20 of these taxa account for approximately 80% of world aquaculture production. Domestication of aquatic species has not proceeded to the same level as it has in the crop and livestock sectors. Genetic biotechnologies must be used both to assist in the further domestication of aquatic species and to help manage and conserve the genetic resources found in wild populations.

Genetic Biotechnologies in the Fishery Sector

3. This could be used, or more widely used, in the fishery sector in developing countries. Genetic biotechnologies that can be used in fisheries and in aquaculture include those that help to manage Background Document provides a summary of recently developed biotechnologies that genetic resources and those for genetic improvement of farmed aquatic organisms.

4. For management of genetic resources, markers can be used in the identification of management units and of endangered species to assist fishery management and they can also help broodstock management in stocking programmes. These markers may be genes, proteins (i.e. the products of genes), sequences of DNA or the phenotypic expression of genes (different colours, shapes etc.). In the 1960's, analysis of proteins revealed a wealth of genetic diversity in wild populations. Protein analysis is now relatively fast and inexpensive, but it requires tissue samples to be stored and transported frozen. DNA analysis is becoming the method of choice because of the very small amount of tissue needed (the DNA can then be amplified in the laboratory by the polymerase chain reaction), the fact that the tissue can be stored dried or in alcohol, and because DNA analysis reveals much moregenetic variation than protein analysis.

5. Several kinds of DNA markers exist, such as RFLPs, AFLPs, RAPDs or microsatellites. These, as well as other kinds of markers, can be used to determine gene frequencies and genetic variation in and between different groups of fish. Studies carried out using these technologies in fish populations have revealed high levels of genetic variation distributed throughout the fish genome.

6. Genetic improvement technologies cover a range of techniques requiring different levels of expertise and resources. Chromosome-set manipulation (i.e. polyploidy induction) is an established technique to increase the number of chromosome-sets (ploidy number) in an organism. Temperature, chemical and pressure shocks applied to fish eggs can be used to produce triploid (3 chromosome-sets) individuals that have desirable culture traits. Sex-reversal and the production of single sex groups of fish is also a simple technology that combines hormone treatment and chromosome-set manipulation.

7. Hybridisation, i.e. the mating of genetically different groups from the same species (intra-specific hybridisation) or from different species (inter-specific hybridisation), is a simple technique that is now easy to accomplish due to our increased knowledge of reproductive biology. A problem is that the gains only occur in the first generation; breeding hybrids with hybrids results in a non-uniform and unpredictable group of fish that is generally not well-suited for culture. Therefore, with hybrids the pure parent-lines must be maintained. The above genetic improvement techniques are considered short-term strategies where the gains are seen in one or two generations.

8. Selective breeding is a longer-term strategy where gains are accumulated at each generation of selection. Molecular markers are now increasing the efficiency of selective breeding by facilitating the identification of quantitative trait loci (QTLs), i.e. genes that control complex characters such as growth rate and environmental tolerance and, subsequently, by making it possible to use molecular markers linked to favourable QTLs to identify desirable individuals or families.

9. Genetic engineering and the production of transgenic organisms is an active area of research and development in aquaculture even though no transgenic animals are on the market. This is a medium-long term strategy in that development and testing of stable transgenic lines requires time. The large size and hardy nature of many fish eggs allows them to be manipulated rather easily and facilitates gene transfer by direct injection of a foreign gene or by electroporation, where an electric field assists gene transfer.

10. In the next three sections, currently available biotechnologies are briefly discussed in the context of fishery management, aquaculture and conservation.

Fishery Management

11. The role that the application of genetic principles can play in the sustainable use and conservation of living aquatic resources is being increasingly appreciated by resource managers, policy makers and the international community. Fishery management requires information on the fishery resources in order to be effective. Primary information needs include:

• an identification of the resource;
• the breeding or stock structure of the resource;
• an estimate of the size of the resource; and
• the identification of key habitat that the resource requires.

12. Genetic analysis of the resources can address these information needs. Gene and genotype frequency data can provide information on, inter alia, species identification, population stock structure, hybridisation and gene flow. Often, data from other sources, e.g. studies of tagged fish or of external characters of fish, cannot provide such information or are extremely difficult to collect in certain areas such as large river systems, floodplains or marine areas.

13. A stock's genetic character can be analysed by examining the frequency of different markers. The use of protein and DNA data in fishery management requires collection of baseline (or background) genetic information. Genetic data were used to determine how sub-groups of Pacific salmon differed from each other in the Pacific Northwest. This required the analysis of hundreds of stocks of salmon but, once completed, endangered stocks were identified, levels of migration were estimated, and the contribution of different stocks to a mixed stock ocean fishery were estimated.

14. Protein and DNA information has been used to identify endangered species that are either inadvertently captured in wild fisheries or that are purposefully taken illegally. DNA analysis of legally sold whale meat revealed that many samples came from protected species of whale and dolphin. Species of shark are often difficult to identify because it is only the fins or flesh that are for sale; DNA analysis can be used to identify the species that provided the tissue and has the added advantage that dried tissue or less than fresh samples from markets can be studied.

Aquaculture

15. Genetic biotechnologies in aquaculture focus primarily on increasing growth rate, but also include increased disease resistance and increased environmental tolerance. There are several biotechnologies that can be applied to farmed aquatic species.

16. Selective breeding, i.e. traditional animal breeding, started with the common carp several thousand years ago. However, it has only recently been applied to a handful of other species of food fish such as catfish, trout and tilapia. Therefore, many farmed aquatic species are very similar to their wild relatives. Selective breeding programmes have yielded significant and consistent gains of 5-20% per generation in species of, inter alia, Atlantic salmon, catfish and tilapia.

17. Chromosome-set manipulation can be used to produce triploid organisms that generally do not channel energy into reproduction because of problems associated with development of reproductive organs. Initially it was thought that this energy saving would result in increased growth rate, but this seems not to be the case. The real advantage of triploids seems to be in their functional sterility (although sterility is not 100%). For example, triploid oysters do not produce gonads and are therefore marketable at times of the year when mature oysters have an off-taste because of gamete production (note, gametes are sex cells - the ovum, or egg (female), and sperm (male).

18. In aquaculture, one sex is often more desirable than the other. For example, female sturgeon produce caviar, male tilapia grow faster than females whereas it is the female trout and salmon that generally grow faster than the males. The production of single sex groups of fish takes advantage of this sexual dimorphism and can be accomplished by manipulation of the developing gametes and embryo. The manipulation can be in the form of DNA denaturisation in gametes followed by chromosome-set manipulation or by hormonal sex-reversal and subsequent breeding. The phenotypic sex of many aquatic species can be changed by administering appropriate hormones. For example, genetically male tilapia can be turned into females through estrogen treatments. These genetic males when mated with normal males produce a group of all-male tilapia that grow faster and have less unwanted matings (that often lead to overcrowding and stunting) than a group of mixed-sex tilapia. Some of the all-male offspring would have two male chromosomes and these could be used as broodstock for subsequent generations, thus avoiding the use of hormones in the broodstock.

19. Hybridization is another simple genetic technology that has become easier with the development of artificial breeding techniques, such as the use of pituitary gland extract and other hormones to initiate gamete development and induce spawning (i.e. the release of fish eggs), and an increased understanding of environmental cues that influence reproduction, such as day length, temperature or water current. Many of the natural reproductive isolating mechanisms that species develop in the wild can now be overcome by fish farmers. Hybridisation can also be used to produce single sex groups of fish when the sex-determining mechanisms in the parental lines are different (for example, hybridisation of Nile tilapia and the blue tilapia).

20. These improvements in reproductive technologies have also assisted aquaculturists greatly in their efforts to domesticate aquatic species. In addition, by making it possible to remove the natural constraints and timing of breeding, farmers are able to mate many more species at the times that are most beneficial, and thus help to ensure a steady and consistent supply of fish to the market.

21. Genetic engineering is a vague term that has come to be nearly synonymous with gene transfer i.e. the production of transgenic fish or genetically modified organisms (GMOs). This technology is progressing rapidly and it is now possible to move genes between distantly related species; a gene that produces an anti-freeze protein has been transferred from the winter flounder to strawberries. Gene transfer in fish has usually involved genes that produce growth hormone and has been shown to increase dramatically growth rate in carp, catfish, salmon, tilapia, mudloach and trout. The same anti-freeze gene that was put into plants was put into salmon in the hopes of extending the farming range of the fish. The gene did not produce enough of the protein to extend the salmon's range into colder waters, but it did allow the salmon to continue growing during cold months when non-transgenic salmon would not grow. Transgenic technology is currently in the research and development stage; to our knowledge there are no transgenic aquatic plants or animals available to the consumer.

Cryopreservation

22. The development of cryopreservation or low-temperature technology allows the short- and long-term storage of gametes. Currently, these low-temperature techniques can only be used on male gametes; eggs and embryos can generally not be stored in this way. Freezing gametes can increase the flexibility of a fish breeder, especially when breeding species where the sexes mature or migrate at different times, when the breeding season is very short, when the breeders are far apart or when one sex is exceptionally rare.

Fish health

23. Genetic biotechnologies are being used to improve fish health through conventional selection for disease resistance and through the use of molecular investigation of pathogens for characterisation and diagnosis. DNA-based technologies are being used now to characterise different species and strains of pathogens. Genetic characterisation of the pathogen may also reveal information about its origin, e.g. DNA analysis revealed two strains of crayfish plague fungus in Sweden: one from the local species and one originating in Turkey. Once the pathogen is characterised, DNA probes can be developed to screen for specific pathogens in tissue, whole animals and even in water and soil samples. These techniques are being used to detect viral diseases of marine shrimp throughout the world and for bacterial and fungal pathogens in fishes in many areas.

24. Genetically engineered vaccines are also being developed to protect fish against pathogens. Genetic immunisation of rainbow trout with a glycoprotein gene from the virus causing viral haemorrhagic septicaemia has recently been shown to induce high levels of protection against the virus. Work is also underway on immunising carp, salmon and other fishes with genetically engineered vaccines for other diseases. Currently, vaccines are questionable in crustacea and may be difficult or costly to use effectively in developing countries.

25. The new molecular techniques are extremely sensitive and can identify pathogens in fish long before there are any clinical signs of the disease. This has implications for quarantine and the trade of aquatic species, which is currently governed by the World Trade Organisation and the Office International des Epizooties. Trade can be restricted based on the disease status of a product or a region; identification of minute quantities of a pathogen or of a new strain of an existing pathogen could change or influence existing trade patterns.

Farming system

26. Appropriateness of genetic biotechnologies must take different farming systems into consideration. Farming systems for aquatic species are diverse and include industrial scale farms, family ponds and culture based fisheries (stocking), in both developed and developing countries. Often, there is a division of the production process where fingerlings or eggs are produced by the seed-supplier, but the grow-out to market size is done elsewhere. In the case of sea going salmon, there is often a seed supplier operating a hatchery near a river, a fingerling producer in a freshwater lake, and another group that grows the fish to market size in the sea. Marine shrimp hatcheries in Asia are usually small family owned ventures, whereas in Latin America they are more industrial in scale. Each different system will have different capabilities to use genetic technologies or genetically improved stocks, and present different impacts on the local environment.

Conservation

27. Genetic biotechnologies can be used to reduce the impacts of farmed fish on wild populations, to identify and manage endangered species and to manage captive populations in aquaria or in species recovery programmes. In several areas, farmed fish must be made triploid, i.e. sterile, in order to reduce their impact on wild populations should they escape from the fish farm. Generally, the planned use of transgenic fish also includes the provision that they are sterile, to reduce the chance of mixing with other fishes. One rational for the production of transgenic fish is that with the savings in production, farms can be located away from environmentally sensitive areas and systems can be recycled (closed). Genetic engineering and polyploidisation can be combined to regenerate threatened species from frozen sperm by denaturing the DNA in an egg of a related species, fertilising with frozen sperm from the threatened species and then carrying out chromosome-set manipulation of the fertilised egg.

Potential areas of research

28. The above sections outline potential areas of research in fishery management, aquaculture and conservation. However, not all research may be appropriate for FAO's mandate to assist developing countries. The question of appropriateness may consider the following elements:

• How does the farming system influence the use of genetic biotechnologies in developing countries ? What is the genotype x environment interaction that results from using different genetically improved strains in different culture systems? Nile tilapia has been greatly improved through selective breeding, however it performs best in medium to high input farming systems that are well managed. If farming systems in developing countries are "simple", can they take advantage of genetically improved species?
• What are the factors that determine or influence the appropriateness of the different biotechnologies in developing countries e.g. their environmental impact; their impact on human health; the status with respect to intellectual property rights; the status with respect to biosafety regulations and controls; the degree of access to the biotechnologies; the level of capacity-building or resources required to use them; their financial cost; their ethical dimension; and their impact on food production and food security. A genetic engineering research project on chinook salmon in New Zealand has been stopped due to a small but vocal group of consumers against all genetic engineering. However, developing countries such as China and Cuba are actively pursuing genetic engineering with little apparent consumer reluctance.
• The relative costs (financial, social, political or otherwise) of the biotechnologies versus the relative benefits (productivity, food security or otherwise). Genetic biotechnologies range in their complexity and in their human and financial resource requirements. Not all developing countries can set up molecular DNA laboratories to characterize genetically their aquatic resources. The marine shrimp farming industry is largely based on wild caught postlarvae; hatchery larvae can not be produced as cheaply during times of the year when wild larvae are available. Therefore, even though reliance on wild larvae promotes disease transfer and prevents genetic improvement, short term economics concerns largely prevail.
• Whether some biotechnologies are more (or less) suited to certain regions or environments (e.g. marginal areas) in the developing world than others. Genetic biotechnologies may allow the range of farmed species to increase by providing wider environmental tolerance. However, this may have biodiversity concerns if the farmed species escape and interact with local species.

29. There are also broad issues on applying genetic biotechnologies that are not specific to fisheries or aquaculture. For example, there is still much basic research needed on the functional importance of DNA variation, i.e. how genetic diversity influences organisms and populations; the blending of genetics and ecology to understand population behaviour in the long (evolutionary time scale) and short term (management time scale). Additionally, research on or the design of information systems dealing with genetic diversity is needed in order to provide better documentation of and access to genetic data in both developed and developing countries.

Suggested Action by the Committee

30. Genetic biotechnologies and Genetically Modified Organisms (GMOs) are very high profile subjects that suffer from misunderstanding and misinformation. There are great potentials, however they must be balanced against environmental risks and cost effectiveness. The committee may wish to consider how to maximize the benefits from these and other new technologies in a cost effective manner without endangering environmental or human health and without alienating consumers. The Committee may also wish to provide advice on how to judge the appropriateness of genetic technologies in developing countries. The Committee is further invited to comment on the prioritisation of the potential areas identified in this document concerning research.