By David N. Leff

Try this trivia question: What color are a mosquito's eyes?

The answer — deep purple — is not all that trivial.

When certain genes that dictate the hue of an insect's orbs are mutated, their eyes are a washed-out white — in effect, albino. This effect, first engineered in the fruit fly, Drosophila melanogaster, in 1982, has led lately to a strategy that may put a severe crimp in the ability of mosquitos to transmit life-threatening diseases.

The key concept, said vector biologist Frank Collins, "is germline transformation brought about by transposons — jumping genes."

Collins, who holds an endowed chair of biology at the University of Notre Dame, in South Bend, Ind., is senior author of a paper in today's issue of the Proceedings of the National Academy of Sciences (PNAS), dated March 31, 1998. Its title: "Stable transformation of the yellow fever mosquito Aedes aegypti, with the Hermes element from the housefly."

"A. aegypti is kind of like a cockroach among mosquitos," Collins said. "It lives in and around human habitation, breeds in small bodies of water — typically discarded cans and tires — and feeds largely on people. What's required for a mosquito to be an important transmitter of pathogens," Collins explained, "is that it has to be biting the human population at a high enough frequency that it can become infected, and then pass the pathogens to other humans."

He added: "A. aegypti is the major transmitter in the world of the Flaviviruses that cause yellow fever [YF] and dengue fever [DF]. YF is pretty much limited to Africa and tropics of the Americas; DF is found largely in Asia and the Americas, but I believe it's also cropping up elsewhere.

"Compared to malaria," he went on, "which is very widespread and pathogenic, YF is relatively limited, and really poses a problem only in the poor parts of the world. There's a very effective vaccine against YF, but it's not readily available or affordable for most populations at risk of the disease.

"For DF, on the other hand," Collins continued, "there is no effective vaccine, and no cure. Once you've got it, you simply have to run the course of the infection — also aptly known as breakbone fever — which can be fatal in many cases."

Collins and his co-senior author, molecular biologist Anthony James, of the University of California at Irvine, have teamed up to test their strategy for committing gene therapy against A. aegypti's pathogenicity.

Versatile Transposons Cut And Paste

"The principle that we've used," Collins recounted, "is essentially the same that worked in Drosophila. It's based on the fact that most of these transposons operate by a mechanism that involves two functional components: One is a region that encodes an enzyme called transposase. It has the ability to snip the transposon out of one location on the genome, then open up a new location and paste it back in.

"The transposable element's second component consists of short inverted repeat sequences, which constitute, in a sense, part of the target of transposase. That enzyme recognizes those inverted repeats, then snips the transposon out.

"Transposase has a second function," Collins continued, "which is to cut open a new location in the genome and effectively reverse the process, pasting the transposon back in."

Collins put together a transformation construct based on the Hermes transposon, isolated from the common housefly (Muscus domesticus). He hooked this sequence up to a functional gene called cinnabar (cn), which normally gives the fruit fly eye its deep red pigmentation. A mosquito with a mutation in this gene doesn't have eye pigment.

"We prepared two plasmids to be microinjected into the mosquito embryos," Collins recounted. "One encoded the transposase. The other contained the two inverted repeats flanking the cinnabar gene. When the enzyme is expressed from the one plasmid, it operationally snips the cn gene and flanking repeats out and gets reinserted, in some cases, into the mosquito genome, thereby providing a sort of wild-type gene, which corrected the mutation."

This cn gene served the co-authors as a marker, visibly reporting that the transposon had moved it from its site in an injected plasmid into a location in the mosquito genome that could nullify the abnormality and rescue the insect from ocular albinism.

Collins then shipped this two-plasmid package off to James, whose lab was poised to microinject it into 920 early-stage mosquito embryos.

"Of 120 adult insects resulting from this germline engineering," the Notre Dame scientist summed up, "60 showed color in their eyes, because of transient rescue. Of the 120, three gave rise to offspring that ultimately formed three independent lines that were transformed permanently, through 10 generations, and still counting."

However, the insect vector for yellow fever and dengue is only a demonstration model of Collins' longer-range target — Anopheles gambiae — the mosquito that spreads malaria. "We're now trying to do the same work with it that we've already completed with A. aegypti," he said.

Shrink-Wrapped Parasites Die In Mosquito Gut

"With A. gambiae," Collins observed, "people have been considering several different genes. My lab is looking at a strain of A. gambiae that has been selected to be refractory to malaria parasites. It actually kills the protozoans by encapsulating them in a melanin and protein matrix.

"But relatively little is known about the molecular mechanism that underlies this encapsulation," he allowed. "We're trying to clone the gene that's responsible for it, with the hope that somewhere in this complex encapsulation pathway there may be something that we can take advantage of.

"We're looking at A. gambiae first," Collins pointed out, "because this is the major malaria vector in the world. But conceivably this strategy could be applied to other malaria mosquitos elsewhere."

He went further: "This particular strategy for genetically transforming the mosquitos, a strategy based on an active transposon, is potentially applicable to most organisms. It's not been used, for example, with vertebrates because there are alternative transformations that don't have to be quite as efficient.

"But a transposon-based technique for transforming something like an insect is something in which you actually have to transform embryo by embryo. You can't produce, for example, a sort of germline cell culture, as you can in vertebrates. Transform one of those cells, then introduce it into an embryo. It just doesn't work with insect eggs.

"But," Collins concluded, "that's what you can do with vertebrates." *