Female silkworm moth on a cocoon. Some transgenic silkworms spin industrial-strength fibers. Photo: Fastily, Creative Commons.
A transgenic organism is any living creature, such as a bacterium, plant, or animal that has received a foreign gene by means of genetic engineering. There are many applications for transgenic organisms in the health and food industries. Scientists have developed a variety of transgenic silkworms that spin industrial-strength or glow-in-the-dark fibers, for example, or make silk with human proteins. A mosquito may soon be engineered to be unable to carry one type of malaria parasite. Field tests are underway to determine the safety of using transgenic pink bollworms, designed to spread sterility to wild bollworms, to protect agricultural crops. Most transgenic research is still in the development phase, and now is the time to address the impact of transgenics on the environment, crop production, and animal and human health.
What are transgenic insects?
Insects are made transgenic when one or more DNA sequences from other organisms are inserted into their genome. The accepted method for making transgenic insects is to splice the DNA intended for insertion into a mobile element, also known as a “jumping gene.” A jumping gene is a segment of DNA that can be integrated at many different sites along a chromosome. In our lab we use a mobile element, called piggyBac, that was discovered by Malcolm Fraser at the University of Notre Dame.1
Interestingly, when the human genome was finally sequenced, many copies of an inactive mobile element from bacteria, calledmariner, were found. Several copies of a piggyBac homolog named looper were also identified. How many other similar genes might be present is not known because they are not easy to identify and characterize; however, one current theory suggests these jumping genes play a role in human evolution.2
Agricultural uses for designer insects
The first application of the new transgenic insect technology is likely to be improvements in the sterile insect technique (SIT). SIT was developed by Edward F. Knipling from peaceful uses of atomic energy. The method employs
- mass rearing of pest insects
- exposing them to damaging radiation at levels that cause sterility
- releasing them over infested areas so that they swamp out reproduction in wild populations
SIT programs have been used for a variety of insects, including Medfly, codling moth, and pink bollworm.3-5 However, some obstacles must be overcome:
- Some insects can’t be mass reared.
- Mass rearing sometimes adds to improved population numbers but at the expense of fitness. (Fitness is the ability of an organism to adapt to its environment.)
- Currently the technique involves sterilization of insects with radiation, which further reduces their fitness.
To make pink bollworms sterile using SIT, the pupal stage is exposed to gamma rays, causing massive chromosome breakage that the cells then attempt to repair by natural mechanisms. The result is the main functions of the cells are retained and the resulting adults are sterile. A side effect of the process is lower competitive mating behavior in the transgenic insects as compared to non-irradiated ones. To compensate for this, very large numbers of SIT insects are released in an attempt to counteract the mating vigor of the wild populations; in pink bollworm SIT, the ratio of sterile to wild insects is 60:1 (by contrast, the ratio is 100:1 in Medfly SIT). The pink bollworm SIT program is ongoing in California (Figure 1).
Map of California and its counties shows the main cotton growing areas (green) and the pink bollworm quarantine zone (red). Pink bollworm is established in the two counties shown in red but not in the large green area in the Central Valey. An SIT program aimed at preventing the spread of pink bollworm from the infested areas into the Central Valley has been in operation since 1969. SIT pink bollworms are shipped daily from mass-rearing facilities in Phoenix, AZ, for release in central California’s uninfested areas.
The next step in controlling pink bollworms is to create a transgenic insect that can breed with, and transfer sterility to, wild insects. How do you breed sterile transgenics? Insertion of a single “conditional” lethal gene into pink bollworms eliminates their susceptibility to radiation’s side effects under certain conditions. Notch is the first conditional lethal gene to be discovered that allows mass rearing of insects. Our lab found the Notch gene in Drosophila melanogaster, the vinegar (fruit) fly, in 1994.6 For an unknown reason the Notch protein works above room temperature to help the embryo develop, but it interferes with embryonic development below room temperature and is therefore conditionally lethal. By mixing a strain of Drosophila melanogaster having two copies of the Notch mutation with an equal number of wild types, we caused the population to collapse in three generations. When we attempted to insert Notch into pink bollworms, however, we ran into the first major problem. Genetic elements that work perfectly well in Drosophila, a fly, do not necessary work the same way or work at all in distantly related insects such as the pink bollworm, a moth. This process is still in development.
The second lethal gene, nipper, also from the vinegar fly and also a developmental gene, was discovered by Luke Alphey of Oxford University, UK. Alphey and his company, Oxitec, UK, made a more elaborate lethal construct that they hope will be used for a number of pest control cases. In transgenic pink bollworms, the lethal effects of nipper are switched off in the presence of the antibiotic, tetracycline, which is used in mass rearing to suppress bacterial infections. When released in the wild, where no tetracycline is available, adult pink bollworms mate and pass on the lethal effect to the next generation of the field population, making them perfect candidates for the new SIT. Alphey and his colleagues at Oxitec are also working to solve the problem of making genes from the vinegar fly work in other insects.7
The problem of transgenic insect fitness
Laboratory rearing of insects naturally selects for traits and behaviors that are not compatible with competitive behavior in the wild, which is one of the dilemmas of the SIT approach. Occasionally lab strains are outcrossed with wild strains to counteract the effects of inbreeding and lack of competition.
The process of making transgenic organisms introduces another form of fitness loss. The act of introducing a new gene, even when inserted by the “natural” mechanism using mobile elements, always leaves the recipient weak, in most cases fatally weak. Of the hundreds of trial insertion events only a few transgenic individuals are able to survive. Fitness is improved by breeding with healthy stocks and then selecting pure strains.
Any attempts to release transgenic insects into the wild would encounter these same issues, that is, reduced fitness compared to wild types because of lab rearing and gene insertion. The first experiment in which transgenic mosquitoes (with fluorescent protein gene markers) were allowed to mix with wild populations in a confined cage study showed the transgenes to naturally decline quickly over time. This is exactly what happens with transgenic pink bollworms. And yet, surprisingly, it took from 4 to 16 generations for any vestiges of the transgenes to finally disappear. I expected reversion to wild type to occur more quickly, and perhaps in the wild it would.8
Designing symbionts for control of plant diseases
Paratransgenesis, another use of transgenic technology in crop protection, is the technique of inserting genes into symbionts that live in host organisms. A symbiont is an organism that depends on another organism (the host) to survive; an example is a clownfish that finds shelter and food in the tentacles of the sea anemone.
In the Chagas disease model, used here to illustrate symbiotic control, the insect vector is a ‘kissing’ or blood-sucking bug that transmits the blood-borne disease from wild and domestic animals to humans; a protozoan is the pathogen and a bacterium is the symbiont (colored circles in the insect’s gut). The first step in symbiotic control is to pick a symbiont that is already present in the vector insect (red circles, step 1 on the drawing). Next, genetically alter the symbiont (purple circles, step 2) to carry a gene product that is lethal to the pathogenic agent. Then insert the altered symbiont back onto the vector insect (step 3) to spread the lethal gene to any protozoans encountered when the inset ingests a blood meal.
Paratransgenesis was pioneered by Frank Richards at Yale University, and our lab borrowed its principles to develop a method to counteract the threat of Pierce’s disease in California.
- The key feature of symbiotic control, which uses symbionts to control pests or disease, is identifying a symbiont that has both an intimate relationship (called mutualism) with a diseased host and access to the pest or pathogen attacking it.
The difference between symbiotic control and biological control, which uses organisms such as parasites or predators to control pests or disease, is the transgenic pest organism acts as a symbiotic control agent itself rather than parasites or predators acting to control the pest.
Symbiotic control is different from ordinary microbial pesticides, which are used the same as commercial insecticides, in having greater selectivity and fewer side effects. The symbiotic agent can be designed to affect only the pathogen causing the given disease (Fig. 2).
Chardonnay grape leaves in a commercial vineyard in Temecula, CA, show signs of Pierce’s disease. This grapevine is near a field plot where a method to control Pierce’s disease is being tested. Note that grapes are still being produced in the presence of the disease. Photo: © 2004, Blake Bextine.
Pierce’s disease is caused by a strain of a bacterium called Xylella fastidiosa (XF). XF originated on the Gulf of Mexico 150 years ago and came to California probably in infected grapevines. Historically, Pierce’s disease occurred on rare occasions in California and disappeared equally quickly because the native leafhoppers are poor at spreading the pathogen.
More recently, in the mid 1980s, the glassy-winged sharpshooter (GWSS), a leafhopper native to southeastern United States where Pierce’s disease is endemic, arrived in California. The new arrival changed everything because it is very efficient at spreading the pathogen that causes Pierce’s disease. Other strains of XF bacterium were also spread such that southern California is now suffering from an epidemic of oleander leaf scorch and similar diseases in ornamental trees.
Symbiotic control is one biotechnology choice for controlling Pierce’s disease. So far in our experiments:
- Carol Lauzon found a symbiont, tentatively identified as Alcaligenes xylosoxidans var. denitrificans (Axd), from the foregut of the glassy-winged sharpshooter10
- David Lampe inserted fluorescent marker genes into the chromosome of Axd (RAxd)11
- Blake Bextine worked out methods of following the movement of symbionts from sharpshooters to host plants12
Confined field trials in commercial vineyards, supported by a permit from the Environmental Protection Agency (EPA), have shown that RAxd:
- does not move into the berries or stems of the developing grapevines
- does not survive in the soil
- is replaced rapidly by non-transgenic wild populations of Axd and other symbionts that are a natural part of the ecosystem
More recently, Lampe produced Axd with a marker gene and a gene product that tested lethal to the pathogen. We’ve called it S1RAxd. Bextine is busy testing the new organism for its ability to cure grapevines of Pierce’s disease and prevent transmission of the pathogen. So far these are all strictly laboratory experiments. We have not received the necessary approval to conduct open field trials. The confined field trials mentioned above were conducted in commercial vineyards with the plants bagged to prevent access by wild insects, and the plants were destroyed at the end of the experiments.
State and federal programs have kept Pierce’s disease muted in southern California so far by quarantine restrictions and by insecticide treatments of GWSS in over-wintering host citrus orchards. The grape and wine industry has signaled that they will not accept a transgenic grapevine solution to this threat, thus taking a major biotechnology tool away. A transgenic insect approach is not practical because GWSS cannot be cultured in large numbers in the laboratory, and classical biological control with parasites and predators can suppress the GWSS but not below the economically tolerable disease threshold of one GWSS per plant. And the grape and wine industry has not yet indicated its position on symbiotic control.
Regulation of transgenic and paratransgenic insects
The Pew Initiative on Food and Biotechnology has called for adoption of strict regulations of genetically modified insects in its January 2004 report.13 The EPA calls the new S1RAxd organism a “microbial pesticide,” and while this application of symbionts is new, there are laws already in place for regulating it.
The major concern with Axd is it is related to a bacterium, if not the same bacterium, that causes nosocomial infections, meaning infections contracted in hospitals, especially, in this case, in the lungs of cystic fibrosis patients. Thus, before any registration would be permitted for a microbial pesticide to be used to control Pierce’s disease in vineyards, this issue would have to be dealt with.
The regulatory process involves science-based risk assessment and uses the public forum as a sounding board and to uncover potential issues the public might have with registration and use. The genetically modified pink bollworm project uncovered an attitude in some individuals and groups that we “should not be doing this,” and this view was officially adopted by the California Fish and Game Commission.
Regulatory officials have to respond to objections to the application of new technologies. They cannot respond to vague misgivings; the law does not allow them to do that. But they can measure and deal with safety issues, such as Axd’s alleged nosocomial property. The use of transgenic insects to improve SIT is seen by many as the least risky test of this new technology because the aim is population collapse rather than the spreading of genes through a population.14
Is man in control of nature or the other way around?
The two agriculture pest problems outlined above, pink bollworm and Pierce’s disease, were both caused by human activities. Initially, pink bollworms, which originated in India, were unknowingly dispersed globally as contaminating larvae in cotton seed shipments. Cotton growers thought it would improve their yields; instead they got a major crop pest. The pathogen causing Pierce’s disease and later the glassy-winged sharpshooter were carried to California inadvertently and now threaten crops and ornamentals alike.
Accidental introduction of the pink bollworm illustrates the fallacy in the common notion that humans are in control of nature. However, recombinant DNA technologies are considered by some to be dabbling with creation.
Certainly, inserting genes into insects or their symbionts raises the issue that genes may move horizontally between species, as well as human safety and ethics concerns. However, the transgenic insects are not fully competitive with the wild type, although they are competitive enough to decrease reproduction of pests. In addition, they lose in competition with wild types and self-destruct in a few weeks. Given that reality, it is hard to understand why these laboratory creations are going to be a problem. You don’t get more vigorous organisms unless you allow selection to occur in nature. Nothing coming out of a laboratory is going to be able to compete with the natural process because an artificial environment lacks full selection criteria.
The fact that transgene fragments can survive as long as they do is somewhat puzzling, but it makes sense if nature has a method of saving spare DNA parts for possible later use. When pest insects develop resistance to insecticides, or when microbes develop resistance to antibiotics, and the pesticides or antibiotics are withdrawn, the organisms revert back to susceptibility. But upon reintroducing the same products later, we find resistance occurs much faster the second time around. This is a form of a population saving “remnants” for later use (in this case insecticide resistance to the first compound) at the DNA level. It is a glimpse of the powerful drive for successful DNA replication, but it suggests we may never be able to completely control a population.
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