IT’S an idea that’s so bold it tends to stop people in their tracks when they first hear it. Why not use the power and precision of genetic engineering to modify wild populations of mosquitoes so that they could no longer transmit malaria? If the scheme works, the impact on public health in the Third World would be enormous.
Like most good ideas, it is fraught with difficulties. But undaunted, many researchers are exploring the notion in detail – and funding bodies such as the WHO, the MacArthur Foundation and the Welcome Trust are backing them. Dissenting voices focus on the technical complications, and say that the idea is diverting resources away from other research aimed at containing malaria.
Malaria is the most prevalent of all the great insect-borne diseases. Every year it kills between one and two million people and causes perhaps as many as 300 to 500 million cases of disease. Four species of Plasmodium, a protozoan parasite, are behind this depressing toll. The disease begins when an infected female mosquito – from one of several species of Anopheles – drinks its fill of human blood. Parasites, carried in the insect’s saliva, tumble into the bloodstream, then head for the liver and multiply, before switching their attention to red blood cells. Symptoms range from chills and fever through to severe anaemia and cruel complications such as cerebral malaria.
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The problem is getting worse. The grand vision of defeating malaria with chemistry has all but faded, confronted with the hard facts of evolution. The parasite has evolved resistance to drugs and proved difficult to tackle by vaccination. The mosquitoes have proved equally resourceful, developing resistance to insecticides such as DDT and avoiding insecticide-treated surfaces. Malaria is a massive problem in Africa, where more than four-fifths of cases occur, but where resources are particularly scarce. And global warming could allow the mosquito and its parasite to spread across a much wider area (This Week, żěè¶ĚĘÓƵ 13 May 1995).
Genetic scheme
Although the battle against malaria needs to be waged on many fronts, there has been an understandable surge of interest in the new genetic technology. Stripped down to its essentials, the scheme goes something like this: first, use genetic engineering to design mosquitoes that cannot carry malaria parasites, and second, send them out into the field, armed with a genetic mechanism that would drive their special genes through natural mosquito populations. “It’s the possibility that we could spread those genes from a small seeding which is the real beauty of present views about genetic manipulation,” says Chris Curtis of the London School of Hygiene and Tropical Medicine, who first suggested similar ideas in the 1960s.
Could it be done? Natural selection has thrown up many species of mosquito that cannot transmit malaria, so there are precedents of a sort. And even in one of the most villainous of all mosquitoes – Anopheles gambiae – research has uncovered a number of strains that cannot carry certain forms of malaria. Understanding the special qualities of these “refractory” strains is an important first step towards the long-range goal.
A decade ago Frank Collins, then at the US National Institutes of Health near Washington DC, and his colleagues bred a refractory strain of A. gambiae. In normal mosquitoes, the parasites mate in the gut, producing ookinetes, which bore through the gut wall and head for the salivary glands. In the refractory mosquitoes these ookinetes emerge wrapped in a tough capsule which effectively stops them in mid-career. The team’s evidence hinted that the making of this capsule was the result of a relatively simple genetic change in the mosquito.
Collins, now at the Centers for Disease Control and Prevention in Atlanta, is currently tracking down the gene, or genes, in question. The search has taken the form of a major effort to identify useful landmarks on the mosquito genome with collaborators Liangbiao Zheng and Fotis Kafatos at the European Molecular Biology Organization, in Heidelberg. “We now have the markers to enable us to map the genetic factors that underlie this phenomenon,” says Collins. As the mapping proceeds – and new results are expected shortly – the team hopes to clone and identify the genes that give the resistant mosquito its special gifts.
As well as studying natural refractoriness, researchers are also exploring the idea of creating it artificially – using genetic engineering to equip mosquitoes with bespoke genes. A number of teams have succeeded in inserting foreign genes into mosquitoes, by injecting them into embryos. But further refinements are needed, says Julian Crampton of the Liverpool School of Tropical Medicine. What is still missing, he explains, is a DNA carrier that could ferry the injected foreign genes efficiently into the embryo’s chromosomes. That would ensure that the new DNA functioned smoothly in the adult insect and was passed down the generations. At the moment, injected genes are taken up haphazardly.
Researchers have solved the problem in fruit flies (Drosophila melanogaster) with the help of a “jumping gene” or transposon called the P element, which occurs naturally in fruit fly populations. Transposons have the extraordinary habit of moving around the genome. Genetic engineers have exploited them to make pieces of DNA that will insert themselves into the chromosomes of fruit fly embryos, even when loaded with a cargo in the shape of a foreign gene.
Several research groups are trying to extend the technique to mosquitoes, but finding a suitable transposon has not been easy. Malaria mosquitoes carry a transposon called mariner, but researchers aren’t yet sure whether it will jump to order. Another message of hope has come with the discovery of the hermes transposon in houseflies. Hermes apparently also works in fruit flies, suggesting that it is no respecter of species barriers. The researchers are now looking to see if it will work in mosquitoes.
No one is playing down the difficulties, but there seems little doubt that mosquitoes will eventually yield to the genetic engineers. Researchers agree that success would bring solid returns and perhaps new ideas for attacking malaria. “My own view is that the major benefits of this technology will come very quickly from using it to analyse how the mosquito and the malaria parasite interact,” says Crampton. The plan is to examine the molecular mechanisms behind that interaction, by adding or deleting specific genes and monitoring their effects.
Another goal is to equip mosquitoes with genes designed to disable the parasite. Studies are already under way in a “model system” in which mosquitoes carrying the malaria parasite Plasmodium berghei infect mice. The research is part of a large collaboration involving Crampton, Robert Sinden of Imperial College, London and Andrea Crisanti of La Sapienza University, Rome.
Ingenious idea
The reasoning behind the project is ingenious. When the malaria parasite is inside its mosquito host, it carries a set of distinctive proteins on its surface. Mice can make antibodies that will stick to these proteins; and a parasite “covered” in such antibodies cannot infect a mosquito. So the plan is to get mosquitoes to generate these antibodies themselves. To achieve this, researchers would have to equip mosquitoes with a gene for making the antibodies, together with control elements that switch on the gene at the right place and time – in the gut, just as the mosquitoes are sitting down for a meal.
The team has already cloned the antibody gene and is on the brink of finding suitable control elements. They are seeking these among the genes that make the mosquito’s digestive enzymes – genes that are switched on in the gut at meal times. “Those have been cloned and sequenced and what the team is doing at the moment is defining the functional parts that control expression of the genes – the promoters,” says Crampton. With these “gene-switches” in hand, the researchers will be able to design an antibody gene that springs into action at just the right time. “Then it will be a question of putting the construct into mosquitoes,” says Crampton. Once again, he stresses that the research is a long way from application. But it hints at what might be achievable in the future – a mosquito that kills its parasite.
If such a plan was ever to move from the lab into the real world, many further refinements would doubtless be necessary. Curtis points out that engineers would probably want to put more than one parasite-blocking mechanism into their transgenic mosquitoes. That would lessen the risk that the parasite would eventually outwit the engineers. And it wouldn’t be enough simply to create large numbers of refractory mosquitoes and release them, hoping that nature would do the rest. Some driving force would be needed to push their genes into wild mosquito populations.
Incredibly, nature provides two potential mechanisms for such a ploy, one of which depends on transposons. Experience with Drosophila suggests that transposons can sweep through natural populations. This is what seems to have happened in the case of the P element, according to Margaret Kidwell of the University of Arizona. If researchers were to introduce a suitable transposon into wild mosquito populations, theory says it would spread. And if it carried a cargo of genes for refractoriness, then those genes should be borne along on the transposon’s coat-tails.
Kidwell and her colleagues are exploring the principle behind this idea in experiments on fruit flies. The research involves monitoring the movement of P elements – loaded with a marker gene – through caged populations in the laboratory. The work has already highlighted the kind of problems that would have to be solved if the idea were ever to be applied in the wild. The link between transposons and their cargo sometimes breaks down if they jump around the genome too much, for example.
Another worry, voiced by some researchers, is the suspicion that the same transposon wouldn’t invade the same mosquito population twice. If for any reason it became necessary to release a second cadre of transgenic mosquitoes – perhaps because the special genes of the first cadre had lost their bite – a different transposon might be needed.
A second idea for engineering wild populations has come out of research on a quite separate – if equally subtle – genetic phenomenon, called cytoplasmic incompatibility. Some insects are infected with symbiotic bacteria (species of Wolbachia) that live within the cells of their sex organs. The bacteria are handed down the generations in the female line, via the cytoplasm of eggs. They are not passed on via sperm.
Within any particular species, some strains may have Wolbachia whereas others don’t. If members of the two strains mate, the bacteria exert an extraordinary influence on the outcome, undermining matings in which they are not passed on. The tendency therefore is for the bacteria to migrate through the population. Nature again provides a precedent. In California, a type of Wolbachia has been caught in the act of rippling through a population of the fruit flies. The infection is spreading at a rate of 100 kilometres per year, according to Michael Turelli of the University of California and Davis and Ary Hoffmann of La Trobe University in Australia.
Some researchers believe that cytoplasmic incompatibility could one day help design wild mosquito populations that don’t carry malaria. “We think it has applied significance, because we could use it as a way to drive genes into populations,” says Scott O’Neill of Yale University. The idea would be to link the genes to a Wolbachia infection in mosquitoes. As the infection spread, so would the genes. O’Neill is weighing up various options, such as putting those genes into an insect virus that is passed on via the cytoplasm of eggs, just like Wolbachia. “Any other element that is coinherited in that cytoplasm will spread with Wolbachia,” explains O’Neill.
“All the data we’ve come up with to date suggest that it looks feasible, but we’re still a long way from putting the whole thing together,” he warns. Anopheles mosquitoes are apparently not naturally infected with the bacteria in question, but O’Neill and his co-workers hope to infect them artificially. The team has successfully transferred a Wolbachia infection from another genus of mosquitoes (Aedes) to fruit flies. “We were quite encouraged by that result to think that we may be able to do the same thing in Anopheles mosquitoes,” says O’Neill.
The team is also trying to find the Wolbachia genes that cause cytoplasmic incompatibility in insects. O’Neill says that if those genes were transferred to mosquito chromosomes – along with a gene for refractoriness to malaria – they would spread through the population, carrying the other gene with them.
Double dose
One theoretical advantage of exploiting Wolbachia as a gene-delivery service is that it could be used more than once. Insects carrying two different strains of the bacteria could invade a population carrying only one strain, argues O’Neill. In theory, this would mean that if something went wrong with a first release of transgenic mosquitoes – if they were outwitted by the parasite, say – a second release could be envisaged.
Everyone agrees that the grand scheme of designer mosquitoes is a long way from being realised. A lot of basic science needs doing both in the laboratory and in the field – on the genetics of wild mosquito populations for instance. What is happening at the moment is that researchers are shaping small pieces of a jigsaw that may, or may not fit together. “We’re imagining strategies that we’re unlikely to be able to implement for at least a decade – or more,” is how Collins puts it.
Should researchers be spending their time on a scheme that may not work? Curtis argues that it is better for gene researchers to hone their skills on malaria rather than on some other more academic problem. An enormous body of knowledge will accumulate, even if the overall scheme remains out of reach.
Ethical worries
But Andrew Spielman of Harvard University maintains that the long-term goal of producing refractory mosquitoes is diverting attention and money away from the less glamorous aim of containing malaria epidemics by more conventional means. He argues that we already have the tools to deal with malaria outbreaks, if we knew when and where to apply them. “I see the real research need now as lying in that direction,” he says. He also believes that genetic manipulation is likely to fail for technical reasons.
If the research was eventually successful, it would certainly raise some difficult ethical questions. Releasing transgenic mosquitoes with the intention of changing wild populations would be a momentous step. “If we are able to put all this together,” says O’Neill, “it needs to be evaluated very carefully to make sure that it does what it’s expected to do. And there’ll have to be a lot of very open public discussion about what is going on.”
Another point is the risk that something might go wrong. After all, the engineered mosquitoes would still bite people, even if they could no longer spread malaria. “Each component will have to be tested very carefully and looked at with critical eyes,” says Curtis. “Being sure that they’re not going to be more effective as a vector of something else is important.”
Malaria kills one to two million children each year, so would some risk be acceptable? “If we had in our hands a way of saving a lot of sickness and death and we turned it down for some clever-clever argument about what it might do – that’s not being quite so ethical as you might think,” says Curtis.