*For offprint requests: tel: (317)494-8032, fax: (317)494-9346, email: bmuir@www.ansc.purdue.edu

Key words: Performance testing, fish, transgenic, risk, fitness components

INTRODUCTION

Overall objectives and methods for this research were presented by Muir et al. (1994). Muir et al. (1995) discussed production of transgenic fish and presented data from preliminary experiments on sexual selection on body size in medaka. In this report, we present potential risks of the evolved transgene and other mechanisms which were found to contribute to risk.

MATERIALS AND METHODS

Risk assessment of fish transformed with growth hormone was assessed by examining four fitness components. Single-cell medaka zygotes were microinjected with one of two constructs containing the human growth hormone gene (hGH) driven by the chicken -actin promoter (cA) or the Atlantic salmon growth hormone (sGH) promoter. Seven cA/hGH transgenic and ten sGlVhGH transgenic founders were identified by PCR amplification of a portion of the hGH gene. Of these 17 founders, one sGlVhGH transmitted the transgene to progeny. The transgene was transmitted at a frequency of 4.3%. In the second backcross generation, transgenic (TR) families were variable in growth characteristics, ranging in body size from 75% above to 8% below that of non-transgenic (POR) conspecifics at 8 weeks of age. Fish with greatest expression failed to reproduce. In the third backcross generation, body weight was measured at bi-weekly intervals to 10 weeks of age.

RESULTS AND DISCUSSION

Prior to 8 weeks of age, TR fish were 17-39% heavier than POR fish. After 8 weeks of age, TR fish did not differ significantly from POR fish in weight. Thus, transgenic fish possessed an early developmental advantage that could increase their reproductive rate. At 12 weeks of age, fecundity and viability differences were examined. TR fish had a 27% fecundity advantage over POR fish, on average, with fecundity increasing with body size more rapidly in TR fish than POR fish. However, viability of TR fry was 69% of that of POR fry. With respect to mating behavior, laboratory experiments were also conducted using natural body size variation of same-aged POR fish. Large males obtained a four-fold advantage in mating success relative to small males. The mating advantage occurred because large males were preferred as mates by females, and because large males could control access to sexually receptive females better than small males could.

The effects of differences in fitness components between TR and POR medaka on transgene frequency and overall population number can be modeled using population genetic theory as shown by Muir and Bell (1987). The effects of introducing 60 adult transgenic fish into a population of 60,000 was examined using a deterministic computer model incorporating sexual, fecundity, developmental, and viability selection, as estimated above. Each effect was first examined independently. Results indicated that 1) with only reduced viability, the transgene was quickly eliminated and presented no risk. 2) With a 25% fecundity advantage, transgene frequency increased and population size expanded. Increases in population size could have negative effects at the community level and hence indicate a potential risk. 3) A 12.5% developmental advantage produced an effect similar to that of enhanced fecundity except that it resulted in a more rapid gene frequency increase and population growth; thus, developmental advantages of transgenics pose a greater potential environmental risk than do fecundity advantages

Results showed that the interaction of these effects could offset each other. For example, the 30% viability reduction combined with a 25% fecundity advantage offset each other, and the transgene was eliminated from the population. But a 30% viability reduction did not offset a 12.5% developmental advantage. The model predicts that this combination should result in an increase in both transgene frequency and population size. Thus, a 12.5% developmental advantage combined with a 25% fecundity advantage and a 30% viability disadvantage, should produce an even more rapid rate of increase of the transgene and population size. Therefore, these transgenic lines would pose a high potential risk to the environment. However, these model results need to be verified in actual populations because some factors that may influence transgene spread and population size of medaka may not have been included in the model, such as density dependent viability, resource availability, and depredation from other species.

Although sexual selection is not expected with the growth hormone construct we used in medaka because the growth advantage dissipated by sexual maturity, similar constructs in other species may maintain a growth advantage after sexual maturity. Therefore, it is important to examine associated risks resulting from sexual selection with other constructs and species. If a 25% size advantage continued to the adult stage in medaka, a 400% mating advantage would have occurred. The effect of this mating advantage would have quickly increased the frequency of the transgene, but would not have had any effect on population size. The altered size of the transgenic fish could have had negative impacts on other species in the community, particularly those with which it interacts ecologically . Thus, any transgene which confers a mating advantage should be considered a risk. A factor which, at first consideration, should decrease risk is the reduced viability of transgenics. However, if developmental and fecundity selection act to increase the frequency of the transgene while viability selection acts to oppose it, a genetic load is imposed on the population which would cause local population extinction, with possible detriments to any other species which depend on the extirpated population for survival.

ACKNOWLEDGMENTS

This research was supported by USDA grant 93-33120-9468.

REFERENCES

Muir, W.M., and Bell, A.E. (1987) Multiple vital functions of the daughterless (da) gene in Drosophila melanogaster and factors influencing its expression. Genetica 72:43-54.

Muir, W.M., Howard, R.D., and Bidwell, C. (1994) Use of multigenerational studies to assess genetic stability, fitness, and competitive ability of transgenic Japanese medaka: I. methodology. In: Levin, Grim, and Angle (eds) Proceedings of the First International Conference on Risk Assessment Methodologies. College Park, MD.

Muir, W.M., Martins, R., Howard, R.D., and Bidwell, C. (1995) Use of multigenerational studies to assess genetic stability, fitness, and competitive ability of transgenic Japanese medaka: II. development of transgenic medaka and mating preferences. In: Levin, Grim, and Angle (eds.) Proceedings of the Second International Conference on Risk Assessment Methodologies. Pensacola, FL.