William M. Muir
Department of Animal Science
Purdue University Information Systems for Biotechnology
November 1, 2001

A concern related to genetically modified (GM, transgenic) organisms is the potential environmental harm if these organisms escape or are released into the environment. Harm can take may take different forms from transient to permanent in time frame and from local to global in scope. Thus, to define harm it is first necessary to distinguish between the terms risk and hazard, which are often confused. In this context, William Muir and Richard Howard (Purdue University, Lafayette, Indiana) define transgene risk as the probability that a transgene will spread into natural populations once released and hazards as the probability of species extinction, displacement, or ecosystem disruption given that the transgene will spread into the population.¹ To show lack of harm from transgenic organisms, either the risk [Risk = P(E) where P(E) represents the Probability that Exposure will occur] or hazard [Hazard = P(H/E) where P(H/E) is the conditional Probability of a resulting Harm (H) given that exposure has occurred] must be close to zero; that is, P(E) 0 or P(H/E) 0. Long-term hazards to the ecosystem are difficult to predict because not all non-target organisms may be identified, species can evolve in response to the hazard, and a nearly infinite number of direct and indirect biotic interactions can occur in nature. Muir and Howard conclude the only way to ensure that there is no harm to the environment is to release only those transgenic organisms whose fitness is such that the transgene will not spread, i.e., P(E) 0, in which case the hazard, P(H/E), is irrelevant because the transgene is lost from the population.¹

In this context, long-term ecological risk can be determined from the probability that an initially rare transgene can spread into the ecosystem. Spread of the transgene into natural populations may result in a number of ways, including 1) vertical gene transfer as a result of matings with feral animals, 2) invasion of new territories as with introduction of an exotic species, and 3) horizontal gene transfer mediated by microbial agents, or a combination of these factors. The relative importance of each factor is dependent on species, transgene inserted, and method used to insert the transgene, respectively.

Vertical Gene Transfer: The first mechanism of spread, vertical gene transfer, is dependent on species modified. Highly domesticated stock developed for poultry, swine, and cattle are not well adapted to the natural setting and may not be able to survive and reproduce there. However, if feral populations are locally available, then local adaptation is not a major barrier to gene spread, as the domesticated GM stock may be able to mate with the highly adapted native populations. Aquatic species present the greatest concern in this regard because aquatic environments are highly connected throughout the world and readily available feral populations exist for all domesticated species. Although feral populations do not exist locally for every domesticated species, if the GM organism has an economic advantage, we must assume that human intervention will transport such organisms to area(s) of the world where native populations exist.

Invasion of New Territories: The second mechanism of spread, invasion of new territories, depends on the functionality of the transgene. The anthropogenic introduction of any exotic organisms into natural communities is a serious ecological concern because exotics could adversely affect communities in many ways, including eliminating populations of other species.² The release of transgenic organisms into natural environments, however, poses additional ecological risks-although transgenic individuals retain most of the characteristics of their wild-type counterparts, they may also possess some novel advantage. A transgene for enhanced environmental adaptation, such as heat tolerance, would allow cold water fish with this gene to invade cool and warm water environments while maintaining populations in current habitats. As such, GM fish could reproduce at a faster rate; their population may increase unchecked and adversely affect other species. As a consequence, transgenic organisms might threaten the survival of wild-type conspecifics as well as other species in a community.³

Horizontal Gene Transfer: The third mechanism of spread, horizontal gene transfer, occurs naturally through viruses and transposons, but at such low rates that it would not normally be an additional concern. However, if a virus or transposon is used to insert the transgene construct, even if the virus is disabled, it may be possible for the element to recombine with other naturally occurring viruses and spread into new hosts.

Regardless of the mechanism of gene spread, the ultimate fate of the transgene will be determined by the same forces that direct evolution, i.e., natural selection acting on fitness. Thus, risk assessment can be accomplished by determining the outcome of natural selection for increased fitness. This conclusion assumes that the natural populations are large enough to recover from such introductions, i.e., natural selection will have time to readjust the population to its previous state. Fitness in this context is not simply survival to market age but all aspects of the organism that result in spread of the transgene. Muir and Howard reduced these aspects to six net fitness components: juvenile and adult viability, age at sexual maturity, female fecundity, male fertility, and mating success.^1,4,5 Mating success is often overlooked because it is not a factor in artificial breeding programs but is often the strongest factor driving natural selection.⁶

Extinction Hazard: Muir and Howard found that pleiotropic effects of transgenes that have antagonistic effects on net fitness components can result in unexpected hazards, such as local extinction of the species containing the transgene.^7,1 Such transgenes were referred to as Trojan Genes. A Trojan Gene is a gene that drives a population to extinction during the process of spread as a result of destructive self-reinforcing cycles of natural selection. For example, if a transgene enhances mating success while reducing juvenile viability, the least fit individuals obtain the majority of the matings while the resulting transgenic offspring do not survive as well. The result is a gradual spiraling down of population size until eventually both wild-type and transgenic genotypes become locally extinct.⁷ These results were later theoretically verified by Hedrick.⁸ Local extinction of a wild-type population from a transgenic release could have cascading, negative effects on the rest of the community.

The interaction of mating success and juvenile viability is not the only mechanism that can produce a Trojan Gene effect. Muir and Howard have shown that there are other ways in which a Trojan Gene can result, such as if the transgene increases male mating success but reduces daily adult viability, or the transgene increases adult viability but reduces male fertility.¹ The latter case is of particular interest because transgenes for disease resistance or stress tolerance can increase offspring viability and transgenes can also reduce male fertility, as has been reported for transgenic tilapia containing the growth hormone (GH) gene.⁹ Extinction hazards predicted in this case parallel the use of sterile males to eradicate pest insects. However, in the latter program, males are completely sterile and must be reintroduced repeatedly to cause extinction. In effect, the viability of sterile males is near 1.0 (due to repeated introduction) while male fertility is 0%. Such population extinction, as a result of the antagonistic pleiotropic effects of transgenes on viability and fertility, represents a new class of Trojan Genes, which suggests that attempts to reduce transgenic male fertility that do not result in complete male sterility may increase hazard rather than reduce it.⁹

Muir and Howard also confirmed that, as expected, if any of the net fitness components are improved by the transgene, while having no adverse side effects, the transgene will invade a population.^1,4 However they showed that advantages in one fitness component can offset disadvantages in another and still result in an invasion risk. Experimental evidence that transgenes have multiple effects on fitness components was presented by Muir and Howard with the Japanese rice fish, medaka (Oryzias latipes).⁴ They found that insertion of a growth hormone gene resulted in a 30% reduction in juvenile viability, a 12.5% reduction in age at sexual maturity, and a 29% increase in female fecundity, relative to wild type. Our model predicted that advantages in both age at sexual maturity and fecundity are sufficient to overcome the viability disadvantage produced by the transgene and would present an invasion risk if released. The model also predicted that for a wide range of parameter values, transgenes could spread in populations despite high juvenile viability costs if transgenes also have sufficiently high positive effects on other fitness components.

This research clearly shows that all six net fitness components must be estimated to determine risk. Simple models, such as those presented by Mclean and Laight that are based on viability or other single fitness components, are very misleading.¹⁰ Also, those components need to be integrated into a model that combines them into one prediction of risk. In the next part, I (W.M.) will examine experiments to estimate net fitness components and review development of the model.

1 | Muir WM and Howard RD. 2001. Environmental risk assessment of transgenic fish with implications for other diploid organisms. Transgene Research. In press.

2 | Bright C. 1996. Understanding the threat of biological invasions. In State of the World 1996: A World Watch Institute report on progress toward a sustainable society, ed. L Starke, 95-113. New York: WW Norton.

3 | Tiedje JM et al. 1989. The planned introduction of genetically engineered organisms: Ecological considerations and recommendations. Ecology 70: 298-315.

4 | Muir WM and Howard RD. 2001. Fitness components and ecological risk of transgenic release: A model using Japanese medaka (Oryzias latipes). American Naturalist 158: 1-16.

5 | Muir WM and Howard RD. 2001. Methods to assess ecological risks of transgenic fish releases. In Genetically engineered organisms: Assessing environmental and human health effects, eds. DK Letourneau and BE Burrows, 355-383. CRC Press.

6 | Hoekstra HE et al. Strength and tempo of directional selection in the wild. PNAS USA 98: 9157-9160.

7 | Muir WM and Howard RD. 1999. Possible ecological risks of transgenic organism release when transgenes affect mating success: Sexual selection and the Trojan Gene hypothesis. PNAS USA 24: 13853-13856.

8 | Hedrick PW. 2001. Invasion of transgenes from salmon or other genetically modified organisms into natural populations. Canadian Journal of Fisheries and Aquatic Sciences 58: 841-844.

9 | Rahman MA and Maclean N. 1999. Growth performance of transgenic tilapia containing an exogenous piscine growth hormone gene. Aquaculture 173: 333-346.

10 | Maclean N and Laight RJ. 2000. Transgenic fish: An evaluation of benefits and risks. Fish and Fisheries 1: 146-172.

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