Genetic engineering, the practice of using molecular tools to insert novel genetic material of particular interest into an organism's genome, is a research enterprise spanning many laboratories globally. This precise form of genetic insertion and manipulation has great potential for custom designing molecular-based solutions to many pest and disease problems that currently appear intractable when confronted with conventional management techniques, such as pesticides, or cultural and biological control practices that aim to eradicate or reduce pest populations to non-damaging levels.

One area of interest for molecular biologists is development of transgenic mosquitoes refractory to transmission of human diseases such as malaria, dengue fever, and yellow fever. Transformation of mosquitoes can be achieved in the laboratory through transposable elements, units of DNA that move from one DNA molecule to another where they insert at random. Transposable elements (e.g., the hermes element from Musca domestica) can be designed to carry marker (e.g., GFP from jellyfish, which glows green under UV light, facilitating rapid identification of transgenic individuals) and strategic genes (i.e., genes that make the transformant behave in the desired way, such as being unable to transmit disease-causing organisms) that are expressed following successful incorporation into the target's genome.

While significant progress is being made in developing transgenic insect technologies and effector genes to transform insect species of interest, very little work has been done with laboratory-generated genotypes to determine the likelihood of transgenic insect establishment, population growth, and persistence when competing with non-transformed wild types in nature. Transgenes conferring fitness advantages could act to promote the spread of particular genotypes while transgenes resulting in fitness costs could be a significant impediment to the establishment and competitiveness of transgenic organisms. Therefore, creating insects with appropriate fitness will be critical to the field-level success of transgenic-based control strategies. While many labs are routinely creating transgenic mosquitoes expressing various novel characters, rigorous assessment of the fitness of genetically engineered mosquitoes is lacking. Fitness of transgenic mosquitoes can be affected by type of transgene inserted, placement of the novel material within DNA and associated mutations or interruption of functional gene sequences, and founder effects resulting from inbreeding between small numbers of transformants when establishing newly transformed lines.

Fitness assessment of transgenic insects is an area of research that needs greater attention immediately if shortcomings associated with transgenesis are to be identified, and mechanisms underlying fitness costs are to be understood and ultimately remedied during the early stages of this emerging technology. University researchers located largely in the United States, England, and Italy, using various laboratory-based studies to investigate fitness of transgenic mosquitoes, are paying closer attention to evaluating fitness of transgenic mosquitoes. One study examined the fate of transgenes in Anopheles stephensi, an important vector of Plasmodium that causes malaria in humans¹. In studies in which caged transgenic mosquitoes could interbreed with non-transformed mosquitoes, the frequency of transgenic alleles declined abruptly and in some instances died out. The mechanisms underlying poor performance were not elucidated but were assumed to be caused by insertion and position effects of novel genes and inbreeding depression of transgenic lines following initial creation.

Another research avenue to investigate fitness was to quantify demographic parameters for three lines of transgenic Aedes aegypti, the vector of yellow fever, and compare them with non-transformed Ae. aegypti parameters. Several factors affect the reproductive output of an insect and ultimately its population size and stability. Important demographic factors are rates of sterility or egg inviability (i.e., progeny production), sex ratio of offspring, juvenile viability, development times, and adult longevity. Workers at the University of California Riverside demonstrated that transgenic Ae. aegypti had a significantly diminished capacity for population increase in comparison to non-transformed Ae. aegypti, and that significant differences existed between lines of transgenic mosquitoes². For example, fecundity was significantly impaired for all transgenic lines in comparison to non-transformed mosquitoes, and one transgenic line produced substantially fewer viable offspring than its non-transgenic counterparts. In this study, the authors demonstrated that negative effects of transgenesis on fitness are not uniform, and a strain that performs poorly in one area may outperform other strains when different characters are measured. For example, impaired fecundity may be correlated with a significantly shorter pre-oviposition period. Data collected from this experiment were used to calculate the intrinsic rate of increase—the rate at which a population can increase when resources are unlimited and predators are not present. This value was calculated for non-transformed and transformed Ae. Aegypti² and inserted into a simple logistic growth equation (Fig. 1). The model began with a population of five individuals and continued for 100 generations with a maximum carrying capacity of 100 mosquitoes in the water container.

Figure 1.

In Fig. 1, non-transformed Ae. aegypti significantly outcompeted all transgenic lines, reaching 50% and 100% of the environmental carrying capacity 47% and 41% faster than transgenics, respectively. Based on their intrinsic rate of increase, non-transformed mosquitoes would be predicted to outcompete transgenic mosquitoes rapidly. The challenge facing molecular biologists now is to create a "super-achiever"—a mosquito that is competitively superior to wild types in nature. This would require building a transgenic mosquito with superior fitness, which would result in a shift of its logistic growth curve to the left of non-transformed mosquitoes (Fig. 1). Development of a "super-achiever" may be possible with use of promoters that drive constructs (i.e., transposable element, marker, and strategic genes) through the target population, ultimately reducing or replacing non-transformed wild type populations.

In contrast to the above study, workers in one laboratory identified no fitness costs associated with mosquito transgenesis³, and in some instances, important traits such as longevity and fecundity were actually enhanced in transgenics in comparison to non-transformed conspecifics. Consequently, it is crucial to develop a comprehensive understanding of the mechanisms driving these conflicting outcomes and for the development of standardized protocols for laboratories investigating fitness-related issues in transgenic insects. Adoption of standardized evaluation techniques would increase our ability to unambiguously quantify and compare fitness of transgenic insects among different laboratories.

Another strategy for controlling pestiferous insects is to use viruses engineered with specific activity towards insects, especially caterpillars. Transgenic baculoviruses, a group of entomopathogenic double-stranded DNA viruses with genetically enhanced toxicity for insects, has been assessed for fitness. These viruses were engineered to express insect-selective toxins produced by spider and scorpion genes and kill more rapidly than non-transformed viruses. Laboratory and field experiments have demonstrated that genetically engineered baculoviruses that express insect-selective toxins have reduced reproductive capacity and rates of transmission. Taken together, these results suggest that engineered baculoviruses are less fit than non-transformed parental wild types and will not likely persist in the environment⁴. It is possible that fitness costs incurred by genetically engineering insects and viruses will have similar and fundamental underpinnings that could be worth elucidating.

Transgenesis is an exciting new technology that promises to revolutionize pest and disease vector control. It is expected to become an important tool in managing many pest problems, most likely complimenting the fields of biological, cultural, and pesticidal control. Like any new emerging technology, there will be initial problems during development, and fitness related issues is just one area among many needing attention. With greater research effort, many of the current problems associated with reduced fitness may be solved once fundamental mechanisms are better understood, thereby allowing the technology to advance to field application.

References

1. Catteruccia F, Godfray HCJ, & Crisanti A. (2003) Impact of genetic manipulation of the fitness of Anopheles stephensi mosquitoes. Science 299, 1225-1227.

2. Irvin N, Hoddle MS, O'Brochta DA, Carey B, & Atkinson PW. (2004) Assessing fitness costs for transgenic Aedes aegypti expressing the GFP marker and transposase genes. PNAS 101, 891-896.

3. Moreira L, Wang J, Collins FH, & Jacobs-Lorena M. (2004) Fitness of anopheline mosquitoes expressing transgenes that inhibit plasmodium development. Genetics 166, 1337-1341.

4. Cory JS. (2000) Assessing the risks of releasing genetically modified virus insecticides: Progress to date. Crop Protection 19, 779-785.

Mark S. Hoddle
Biological Control Specialist
University of California, Riverside