Scientists Refining Methods For Genetically Altering Insects

FROM LAB TO FIELD: Florida's Marjorie Hoy holds a dish containing several hundred Western predatory mites, which she transformed by injecting a plasmid containing a gene directly into females' ovaries. Creating transgenic insects is seen by some scientists as a new way to manage and possibly eradicate enduring problems such as malaria and agricultural damage from the Mediterranean fruit fly. Researchers appear to be making significant strides in refining the technique. Meanwhile, critics are raising concerns about genetically modifying insects and the implications of adding new genes to these organisms in the wild. One as-yet-unpublished study used genetic technology to alter mosquitoes. A team of researchers from the University of Notre Dame; the University of California, Irvine; and the University of Maryland has successfully transformed the Aedes aegypti mosquito with a transposable element, a small unit of DNA that can move from one part of a genome to another, called Hermes. The experiment is a starting point for creating mosquitoes genetically incapable of carrying the parasite responsible for malaria, which kills between 1.5 million and 2.7 million people annually, according to the World Health Organization. The success of the technology used in the study will let scientists pose specific questions that could offer a new approach to control the transmission of plasmodium parasites responsible for malaria in humans. Though there are four such parasites, P. falciparum is the most virulent and causes the most illness and death. THE LONG VIEW: Notre Dame's Frank Collins maintains that controlling malaria with transgenic technology will not be viable for at least a decade. "Now we can begin to ask the question: 'Can we block parasite development by introducing a particular gene into the genome?'," notes senior investigator Frank H. Collins, Clark Professor of Biology at the University of Notre Dame and former chief of the entomology branch at the Centers for Disease Control and Prevention's Division of Parasitic Diseases. NO BOOM: Maryland's David O'Brochta doesn't anticipate that the technology will lead to great corporate growth. The researchers used a transposable element called Hermes, which was isolated from the housefly by David A. O'Brochta, an associate professor at the University of Maryland Biotechnology Institute (D.A. O'Brochta et al., Genetics, 142:907-14, 1996), to rescue or correct a mutation in the eyes of the mosquito. The normal, or wild-type, gene was inserted into the transposable element, or transposon. The element and the wild-type gene were put into plasmids, extrachromosmal DNA, which were injected into mosquito embryos. The wild-type gene was found in 8 percent of the embryos' offspring (the so-called G1 generation), meaning it was incorporated into the insects' germ line, or reproductive system. While Aedes aegypti is not a vector for human malaria, it does carry the parasite responsible for an avian form of the disease, so it offers a laboratory model in which attempts to block the parasite can be made, Collins explains. He and his colleagues are also planning similar research with the mosquito Anopheles gambiae, the strain responsible for the transmission of most human malaria in Africa. While the new technology has potential, it does not necessarily translate into anything like a boom in jobs, researchers caution. "I don't anticipate this technology to spawn a tremendous growth in the private sector," except for using the technology to potentially make better silkworms and honeybees, comments O'Brochta. TIMING IS KEY: Wisconsin's Bruce Christensen notes that global concern about infectious and parasitic disease is helping to drive transgenic insect research. But the technology does come along at a time of concern about global public health problems, which may create research opportunities, according to Bruce Christensen, professor and chairman of the animal health and biomedical science department at the University of Wisconsin, Madison, and program director of the university's National Institutes of Health-funded Tropical Disease Research Unit. "It is a good time from the standpoint that everyone now accepts the fact that emerging and reemerging infectious and parasitic diseases are a very serious threat and perhaps may be our most important challenge in public health worldwide." Photo: Jim Gathany MUTANT MOSQUITO: A wild-type ITALICS Anopheles gambiae END ITALICS is shown next to an Anopheles gambiae mosquito that is homozygous for a mutant form of the white gene, which controls eye color. "Controlling malaria with transgenic technology is very much a strategy for the future and is unlikely to be applied for at least a decade or more," Collins notes. An overall strategy has several components. Using transgenic mosquitoes involves identifying genes that could block parasite development, genetically engineering these genes into mosquitoes, and "driving" a gene or genes into the natural population. While Hermes might be a potential mechanism to do that, Collins acknowledges there are no candidate genes that can block parasite development. "There are candidate mechanisms for which there are genetic bases, which point the way toward identifying possible genes." For instance, he has developed a strain of A. gambiae that encapsulates the malarial parasite in melanin and kills it. The process is determined by one major gene, plus some minor ones, he says. But those genes are not yet identified. UNSUSTAINABLE? Harvard's Andrew Spielman contends that malaria control by transgenic mosquitoes ultimately cannot succeed. Malaria control by transgenic mosquitoes, however, is really a doomed strategy in the view of Andrew Spielman, a professor of tropical medicine at Harvard School of Public Health. Though acknowledging that transgenic research can produce volumes of important basic information on the interaction between the mosquito host and the malaria parasite, he argues that "such a genetically engineered mosquito could not be deployed if it continues to annoy people and subsequently requires nurturing. "Furthermore," he adds, "such a malaria prevention effort could not be sustained because the refractory gene would not remain linked to the genetic construct that was used to drive it through the vector population. An initially successful antimalaria intervention that ultimately fails will burden human health because the residents of the intervention site will have lost their preexisting immunity." "I agree with [Spielman] that if you put all your eggs in this basket, you're really going to be disappointed," says Christensen, who has coauthored an unpublished study using Hermes to transform Aedes aegypti. But understanding gene structure and function in mosquitoes can produce new control strategies, Christensen contends. Photo: Jim Gathany FEEDING FRENZY: The mosquito Anopheles gambiae takes a blood meal. Collins sees the possibility of using several different genes successively or simultaneously in an attempt to thwart the possibility that the parasite will get around the strategy. "This is never going to be the 'silver bullet' fix," he says. The potential transgenic strategy to control malaria or study host-parasite interactions is supported by more than a decade of basic research aimed at isolating and using transposable elements for shifting desired genes in insects. The isolation and use of the P element in Drosophila (G.M. Rubin, A.C. Spradling, Science, 218:341-7, 1982) was a key stimulus in the search. But it was the seminal work of Barbara McClintock, who in the 1940s and 1950s discovered that such elements exist, that is the ancestor of this modern work. McClintock earned the 1983 Nobel Prize for physiology or medicine. Scientists found, however, that the P element could not transform other insects. The precise explanation is uncertain, notes Hugh Robertson, an associate professor of entomology at the University of Illinois. One reason appears to be the presence of a particular protein in Drosophila that seems to be necessary for the P element to work. "It's possible that even though other insects have homologous proteins, the P element doesn't interact properly with it. But that's not been proven," he says. TRANSGENIC MITE: An adult female predatory mite, studied by Florida's Marjorie Hoy, feeds on a two-spotted spider mite, a major pest of agriculture and ornamental plants. Consequently, researchers have focused on finding other transposable elements. Aside from Hermes, others have been isolated, for instance Mariner from another form of Drosophila, D. mauritiania (D.S. Haymer, J.L. Marsh, Developmental Genetics, 6:281-91, 1986), and Minos from Drosophila hydei (G. Franz, C. Savakis, Nucleic Acids Research, 19:6646, 1991). Chalambos Savakis, a researcher at the Institute of Molecular Biology and Biotechnology in Crete, and his colleagues used Minos to transform the medfly (T.G. Loukeris et al., Science, 270:2002-5, 1995). They successfully rescued white-eyed mutants using the wild-type eye color gene. University of Maryland Center for Agricultural Biotechnology 5115 Plant Sciences Bldg. University of Maryland College Park, Md. 20742-4450 (301) 405-1581 Fax: (301) 314-9075 http://www.umbi.umd.edu/~cab/insect.html Transgenic Arthropod Team Animal and Plant Health Inspection Service U.S. Department of Agriculture 4700 River Rd. Unit 147 Riverdale, Md. 20737-1237 (301) 734-5394 Fax: (301) 734-8669 http://www.aphis.usda.gov/biotech/arthropod Tropical Disease Research Unit 1655 Linden Dr. University of Wisconsin-Madison Madison, Wis. 53706 (608) 262-3177 Fax: (608) 262-7420 Union of Concerned Scientists 1616 P St., N.W., Suite 310 Washington, D.C. 20036 (202) 332-0900 Fax: (202) 332-0905 http://www.ucsusa.org Transposable elements between approximately 1,200 and 3,000 bases long, about enough to encode one protein, O'Brochta points out. The protein is called a transposase, and it is essential for the element to be integrated into the genome, he says. At either end of the element are "inverted repeats"-a series of bases, for example 12 in Hermes-that are in the opposite orientation from one another. "Those inverted repeats define the limits of the elements for us," explains Robertson. Transposable elements are not the only method used in such research. Marjorie Hoy, a professor of entomology/nematology at the University of Florida, has transformed Western predatory mites (not insects because they hav four pairs of legs instead of three and lack wings) by injecting a plasmid containing a gene directly into the ovaries of females. The gene becomes integrated into the egg by an unknown mechanism, notes Hoy. The mites prey on other mites, which are agricultural pests. In an eight-week field test in Florida, the mites failed to flourish as hoped. Yet Hoy and others regard the test as successful. "It was the first attempt," says O'Brochta. "She forged the way. Just the fact she's gone through all the steps required to take a transgenic insect from the laboratory to the field is probably the most significant aspect of that work." In Crete, Savakis is working to improve medfly control by using transgenic technology in refining the sterile insect technique to produce only males. The present technique produces equal numbers of sterile males and females. Only fertile female medflies damage fruit by laying their eggs in it. Even if they are sterile, they will damage fruit by injecting their ovipositors in an attempt to lay eggs. That transmits bacteria and fungi. They also decoy sterile males away from fertile females. HOUSEFLIES: The fly with creamy-white eyes is a mutant lacking eye pigment; the fly with red eyes is a nonmutant fly with normal eye pigmentation; the fly with apricot-colored eyes sitting on top of the tube is a mutant fly in which the gene for the defective enzyme has been added and expressed. Savakis is working to develop transgenic technology to solve that problem by producing only sterile males. He's had several ideas he wants to investigate. One is turning females into males by using the transposable element to insert genes, causing a sex transformation. Another possibility is to manipulate the genome to protect males, but not females, from an environmental stress such as heat or heavy metals in the insects food. "Such schemes are limited only by our imagination and knowledge of genes," he says. And, at least in the United States, by government review and regulations. Field tests of genetically altered organisms must be approved by the U.S. Department of Agriculture (USDA). Once a formal proposal is submitted, the department has 120 days to review and approve it, according to Orrey P. Young, the team leader in USDA's Animal and Plant Health Inspection Service. Young is in charge of evaluating and approving proposed field releases of transgenic insects and arthropods. He advises researchers who are planning field tests to consult with his office to determine what experiments and analyses need to be done to support an application. "There needs to be regulation. Not many people are comfortable with the idea of putting new genes in insects," says Margaret Mellon, director of the Agriculture and Biotechnology Program of the Union of Concerned Scientists in Washington, D.C. Insects can quickly multiply, she notes. "There's reason to be concerned about this new and not-well-understood technology." Harvey Black is a freelance science writer based in Madison, Wis.

Scientists Refining Methods For Genetically Altering Insects

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