Australasian Biotechnology (backfiles) AusBiotech ISSN: 1036-7128 Vol. 8, Num. 4, 1998

Conference Paper

Robert H. Devlin

Fisheries and Oceans Canada, West Vancouver Laboratory, 4160 Marine Drive, West Vancouver, B.C., Canada V7V 1N6Ph: 1-(604)-666-7926; Fax: 1-(604)-666-3497; e-mail: devlinr@dfo-mpo.gc.ca

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The production of finfish such as salmon in intensive marine aquaculture requires the input of expensive high-energy feeds to allow maximization of growth rates and thus economic return for the producer. In addition, faced with high initial capital startup costs and a myriad of engineering challenges, biological impacts (e.g. disease) and intensive competition among international producers, salmon farmers now must optimize production efficiency to a high level to survive in the marketplace. Improvements in efficiency are being achieved through a number of strategies, including enhancement of nutrition and husbandry procedures, disease therapeutics, and the application of biotechnologies such as monosex culture, sterilization, vaccine development and disease diagnostics (Donaldson and Devlin 1996). The production of transgenic aquatic organisms also holds great promise for enhancing aquaculture in the future, but many challenges remain, including optimizing phenotypes, potential ecological impacts, public perception, and consumer acceptance.

Transgenesis in Animal Species

The past decade has seen an explosion of research on genetic engineering of animals, and aside from model genetic systems (mice, Drosophila, etc.), fish have been one of the most intensively studied systems to date (see reviews: Gong and Hew, 1995; Hackett 1993). This effort was largely stimulated by experiments in the early 1980's where transgenic mice containing growth hormone gene constructs were shown to be capable of growth enhancement (Palmiter et al. 1982). This potential, when extrapolated to agricultural and aquacultural species, has important implications for global food production. However, transgenic mammalian agricultural species (e.g. pigs) have not shown major improvements in growth rate (see Pursel 1989) thus far, perhaps due to their extensive history of domestication and selection which has already made enormous gains and makes further enhancement more difficult. In contrast, some fish species containing growth hormone gene constructs have shown dramatic enhancement of growth (see below). Many other production traits other than growth are also amenable to influence by transgenesis in fish, for example the control of flesh quality and character (e.g. lipid content or type), disease resistance (e.g. expression of peptide antibiotic genes, or somatic transgenic approaches using DNA vaccines), or control of reproductive characteristics (e.g. maturation or sterilization).

Transgenesis in Fish

DNA constructs have been delivered to fish cells and embryos using a variety of methods including microinjection of eggs (Fletcher and Davies, 1991), electroporation of sperm and eggs (Symonds et al. 1994; Zhao et al. 1993), retroviruses (Lin et al. 1994), and particle bombardment (Zelenin et al. 1991). Microinjection has been the most common method employed, and provides a simple method for producing germ-line transgenic animals (e.g. Chourrout et al. 1986; Dunham et al. 1987; Fletcher et al. 1988; Devlin et al. 1994). Typical frequencies of gene retention after microinjection of salmonids are 1-10%.

Transferred DNA is often inserted into the host genome after one or two embryonic cell divisions, resulting in mosaic founder individuals and lower than expected ratios of transgene segregation to F1 progeny. Transgenes are stably inserted into the host genome in random concatemeric forms (Tewari et al. 1992; Devlin et al. 1995a), and are transmitted to F2 and subsequent generations in a Mendelian fashion (Shears et al. 1991) as dominant genetic traits.

Several promoters have been examined for their abilities to express genes in fish cells, although the majority of the information is derived from transient expression studies. Non-fish promoters (mMT, SV40, CMV-IE, RSV, chicken B-actin, hs70) and fish promoters (salmon MT, flounder and pout antifreeze, carp B-actin) are active in fish cells (see Devlin 1996), suggesting that considerable flexibility may exist for regulatory element choice for transgenic fish studies (Friedenreich and Schartl, 1990).

A wide range of expression levels and effects on phenotype are often obtained depending on the chromosomal site of insertion (Tewari et al. 1992; Du et al. 1992; Devlin et al. 1994). The variable influence of a particular construct in different tranagenic lines can be very useful when selecting appropriate phenotypic characteristics resulting from a transgene, particularly since we only have a rudimentary ability to predict the expression level and phenotypic consequence of a particular regulatory element/structural gene combination. Vectors are now being produced with border elements that may reduce chromosomal position effects on trnagene expression and may provide more predictable expression patterns and levels when this is desired (Caldovic and Hackett 1995).

Growth Enhancement in Transgenic Fish

The first studies examining transgenesis in fish utilized mammalian growth hormone gene constructs, and while some reports indicated growth enhancement (Zhu et al. 1985), some species (e.g. rainbow trout) were not stimulated by the same constructs (Guyomard et al. 1989, Penman et al. 1991). This was puzzling since it is known that the promoters used in these studies are active in fish cells, and that purified GH protein is capable of stimulating growth of many fish species (including rainbow trout) after intraperitoneal injection (McLean and Donaldson, 1993). However, subsequent studies showed that mammalian genes may have difficulty in expressing at high levels in fish cells due to improper transcript processing (Bearzotti et al. 1992; Bétancourt et al. 1993) suggesting that the use of homologous gene constructs may be more suitable. Gene constructs that contain piscine rather than mammalian GH coding regions but are still under the control of mammalian or viral regulatory elements are active and capable of stimulating growth by as much as two fold relative to nontransgenic controls (Dunham et al. 1992; Chen et al. 1993; Martinez et al. 1996).

Gene constructs have now been developed that are comprised entirely of fish DNA sequences, and these have been tested for their effects in salmonid species. An "all-fish" construct (opAFPGHc) comprised of the ocean pout antifreeze promoter fused to a chinook salmon GH cDNA (Du et al. 1992), results after approximately one year of growth in a three- to ten-fold increase in size of transgenic Atlantic, coho, and chinook salmon, as well as rainbow and cutthroat trout (Du et al. 1992; Devlin et al. 1995a and 1995b).

Similarly, an "all-salmon" gene construct (OnMTGH1) has been produced that is comprised of the sockeye salmon metallothionein-B promoter fused to the type-1 GH gene from the same species (Chan and Devlin, 1993; Devlin 1993); this construct is identical in concept to that originally used to stimulate growth in transgenic mice. The OnMTGH1 construct stimulates growth in transgenic salmonids (Figure 1) by approximately 11 fold relative to controls (Devlin et al. 1994), and induces precocious smoltification (see below). Individuals transgenic for OnMTGH1 have enhanced grow rate, but they do not become excessively large, growing only to approximately normal adult size; however, their accelerated growth rates allow them to achieve sexual maturity in two rather than four years.

Figure 1. Control and transgenic coho salmon containing the OnMTGH1 gene construct at 14 months of age.

The most rapidly growing transgenic individuals can display some morphological disruptions in the cranium (Devlin et al. 1995b) analogous to acromegaly syndromes observed in mammalian systems overexpressing GH. These morphological disruptions can become quite severe and can ultimately affect respiration, feeding, viability and growth rate. However, individuals that initially display intermediate growth stimulation (arising from lower expression either by the use of a weaker promoter or due to transgene insertion characteristics) have a normal appearance and ultimately become the largest and most viable individuals. We have achieved appropriate growth rates for transgenic salmonids by two methods: 1) selection of strains with weaker growth stimulation presumably arising from transgene insertion characteristics, and 2) by the use of constructs regulated by a weaker promoter.

The growth effects observed with the opAFPGHc and OnMTGH1 gene constructs in transgenic salmonids are much more pronounced than those observed in other fish species. These results suggest that either gene constructs derived from piscine sequences may function better in transgenic fish, or that salmonids may possess a greater capacity for growth stimulation than other fish species (Devlin 1996). Salmonids possess a remarkable capability for growth enhancement through treatment with somatotropins, and this may be due in part to their life history characteristics. Compared to other cultured fish species (such as carp and tilapia), salmonids display relatively slow natural growth rates that are further reduced by seasonal depression or cessation of growth during the cold winter months. This strategy for overwintering in cold riverine environments with limited food resources undoubtabley enhances survival in the wild, but the consequent growth rates can be less than desirable for aquaculture production applications.

Anadromous salmonids naturally display enhanced growth rates after undergoing a spring transformation from fresh water juveniles into smolts (Clarke et al. 1986). Smotification is a physiological adaptation that allows salmonids to osmoregulate in sea water, and is associated with increases in circulating GH levels (Sweeting et al. 1985). Treatment of salmonids with somatotropin protein preparations can induce precocious smoltification (Shrimpton et al. 1994), an effect that is also observed in GH transgenic salmonids. Thus, the growth enhancement effects in transgenic salmonids may be mediated by a dual action of GH, directly enhancing growth and also transforming fish into smolts that have a naturally higher capacity growth.

For other fish species, growth acceleration may be much more difficult to achieve where natural growth rates are high and physiological systems other than endocrine levels of GH may be regulating growth. It is thus recommended that physiological and growth studies examining the response to purified GH protein be conducted prior to embarking on lengthy and costly transgenic programs.

Effect of the Laboratory Environment on Performance of Transgenic Organisms

The results of the growth enhancement studies described above suggest great promise for improving production efficiencies in aquaculture. However, it is important to note that this information has been collected primarily under laboratory conditions that may not be ideal for maximal growth rates. Thus, to some degree, the growth stimulatory effects may be due to an interaction between the new transgenic genotype and the less-than-ideal laboratory environment. Thus, to determine the true potential of transgenic organisms for aquaculture, it will be necessary to examine production performance in situations that approximate production conditions where growth rates are normally maximized (see regulatory issues below). In addition, for growth-enhanced fish, commercial suitability will depend not only on growth rate, but also on many additional factors including feed conversion efficiencies, disease resistance, flesh quality, as well as on consumer acceptance of the genetically-engineered fish.

Regulatory Control of Transgenic Fish in Aquaculture

Considerable concern exists regarding the implementation of transgenic salmonids into aquaculture production systems (Kapuscinski and Hallerman 1991), and this concern is primarily associated with the potential for the escape of production animals. For open ocean net pens, such escapes occur due to nautical accidents, extreme weather, or animal handling mistakes on the farm. In ocean situations, or in other large bodies of water or rivers, it is very unlikely that escaped animals could ever be recaptured. Escaped transgenic fish have the potential to have direct impacts through competition with other members of the ecosystem, but these effects are likely to be of a short duration (single generation) and, if not frequent, are likely to have minimal impacts unless large numbers of animals have escaped. However, it is the reproductive interaction between transgenic organisms and wild members of the same species which has generated the greatest concern due to the potential perpetuation of impacts and because of potential long-term genetic impacts on conspecifics in wild populations. Despite these concerns, almost no data exists to indicate whether transgenic fish pose an actual risk to the natural environment, and regulations and public and academic concern have developed largely as a risk-averse reaction to potential impacts.

Empirical study of transgenic animal model systems may allow determination of the true benefits and potential risks that are associated with transgenic approaches to improvement of aquacultured species. The fitness of transgenic organisms relative to their wild counterparts will determine whether a transgene persists in nature, and this fitness will depend on the myriad of phenotypic characters that a transgene may influence (Devlin and Donaldson 1992). For example, growth-enhanced transgenic salmonids have enhanced appetites that could provide a foraging advantage (unpublished data), but also have impaired swimming ability (Farrell et al. 1996) that could impact on the capture of prey, escape from predators, or ability to successfully navigate spawning streams.

In practice, it may prove difficult to experimentally determine whether transgenic organisms pose a risk to natural ecosystems. Consequently, containment methods need to be developed that will reduce the probability that escaped transgenic fish could reproductively interact in nature (Devlin and Donaldson 1992). Physical containment systems such as secure sea pens or land-based production facilities can be coupled with biological controls such as monosex culture in areas where indigenous populations do not exist, or induced sterility (e.g. hybrid infertility, triploidy, or gene constructs designed to eliminate reproductive function) where conspecifics are found.

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