The perfect swarm: GM mosquitoes

In a business park just outside Oxford, scientists are carefully breeding millions upon millions of mosquitoes. It’s not that they are particularly fond of these biting insects. The opposite, in fact, as their mosquitoes belong to a genetically engineered strain that could help tame the rampant spread of Dengue fever around the world.

I have the cover story in this week’s New Scientist that examines the science behind these GM mozzies and explores the potential of these and other genetically engineered species for controlling agricultural pests or invasive species.

The idea is based on the so-called “sterile insect technique” (SIT) and is brilliantly simple: you breed up vast numbers of your target pest in the laboratory, render them sterile and let them loose in the environment. Provided these infertile imposters mate with their wild confreres (and not just with each other), a whole load of barren eggs should bring the population close to collapse.

Since its first deployment in the 1950s, SIT has been a powerful tool for the management of insect pests, used to control the Mediterranean fruit fly across large areas of Latin America, the codling moth in Canada and the new world screwworm from the United States, Central America and North Africa to name but a few examples.

Now, with the help of cutting-edge molecular techniques, SIT is getting a 21st century makeover. Rather than relying on radiation to induce sterility as has been the case in most SIT campaigns in the past, it is now possible to use genetic engineering to achieve the same result.

In May, I paid a visit to Oxitec, the biotech company in question, and interviewed its founder and chief scientist Luke Alphey at length. In the interests of transparency, and because Dr Alphey said a lot of interesting things that I was not able to include in my feature, here is a cleaned-up transcript of our conversation:

Henry Nicholls: What are the drawbacks of using radiation to sterilise insects?

Luke Alphey: The sterilising dose of radiation is going to be bad for the insects and the extent to which that makes them sickly or short-lived or less competitive, specifically less able to find and compete for mates in the field, [matters a lot]. You have to release more insects to compensate for that. If that’s a small effect it’s no problem. If it’s a big effect it’s a huge problem.

HN: Are mosquitoes more troublesome in this regard than other insects?

LA: Yes. It varies from one species to another. It has been a problem for mosquitoes to find a sterilising radiation dose that doesn’t weaken them too much.

HN: Do we know why that is?

LA: I don’t. The SIT programmes that have worked have all found a sterilising dose that isn’t too bad. You want to do it just before you release them. Radiation is much worse on proliferating cells than on non-proliferating ones. So ideally to minimise the damage, you would irradiate the adults but they are much harder to handle than pupae. Mosquitoes are all spindly so that if you pack them into any kind of space, they all tangle up and you sort of end up with legs and wings and a lot of physical damage. You can irradiate pupae but that gives you not-very-happy adults. So the net effect is that there hasn’t been large-scale radiation-based SIT for mosquitoes. Chemical sterilisation worked moderately well in the 1970s using mutagenic chemicals.

HN: So a genetic approach to sterilisation bypasses all these problems. But it’s also an opportunity to introduce far more subtle consequences for the organism isn’t it?

LA: Exactly. You can further refine the phenotype you want. With irradiation you inevitably get sperm with damaged DNA. Eggs die before hatching. One of the things we can do with the mosquitoes is arrange that they die later in development rather than earlier. This is quite important for container-breeding mosquitoes like Aedes aegypti [the main vector for Dengue Fever]. They are breeding in pretty clean pools of water – rainwater, human-stored drinking water that sort of thing. So the larvae are competing for food in this little pool. If you were to arrange for some of them to die as embryos then the others would have more food and the larvae tend to do better.

HN: So your intervention could be counter-produtive?

LA: It could, in theory. Mathematical models say it could. If there’s strong density-dependent competition, eliminating some of them as embryos could actually mean you get more out than you would have done before.

HN: So why did you settle on Aedes aegypti as the target species as opposed to, say, the Anopheles species, which transmit malaria?

LA: There’s a whole series of reasons. Biological, sociological, economic.

1. Dengue is transmitted really only by this mosquito. There are one or two minor secondary vectors in some parts of the world of which the Asian tiger mosquito Aedes albopictus is the best known. It’s debatable whether that can sustain an epidemic at all. It’s certainly epidemiologically not significant compared to A. aegypti. So basically around the world you’ve got one mosquito transmitting this disease and our technology is exquisitely species-specific. So the sterile males that are released will only court and mate with wild females of the same species and not even with other species of mosquito let alone with bees and butterflies and whatever it is in the environment. So from an environmental point of view that’s extremely attractive. But if the particular thing you want to deal with [like malaria] is [transmitted by] a dozen different species that’s obviously more of a problem. So with Dengue, where it’s clear that in every epidemiologically relevant setting it’s this one mosquito that’s the problem that’s very attractive when you’re thinking about a species-specific technology.
2. Dengue is not as big a disease as malaria but it is a huge problem – there are 50 to 100 million cases a year and unusually for an infectious disease it is increasing in prevalence and severity around the world. There are no drugs and no vaccine. There is nothing apart from mosquito control and there aren’t many tools for that. So the universe of Dengue-control technologies is pretty simple and where we would fit into that is pretty clear. With malaria, there are more options. Drugs, ACT therapy, bed-nets. There’s a wider range of things you might do for malaria. The upshot of that is that our technology will be very, very widely applicable for Dengue. I think it will be beneficial in just about every major transmission setting you can think of. For malaria, I think it’ll be one of the tools in a larger arsenal. It’ll turn out to be extremely valuable in some areas, a piece of the jigsaw puzzle in other areas and perhaps not that relevant in other areas.
3. It’s important to release only males. First, only females bite and males don’t so you can release lots of males and they won’t do any harm, they won’t transmit disease, they won’t bite people. They are harmless. Whereas females – even sterile ones – would bite and would potentially transmit disease. So you don’t want to release sterile females if you can avoid it. Second, if you release lots of sterile males along with lots of sterile females then the sterile males that you released will hang around and court the sterile females you released alongside them. And they won’t so enthusiastically – to anthropomorphise for a moment – go out and look for the wild females. Those sterile females are effectively distracting sterile males from doing what you want them to do. So they are not even neutral to the program but actually negative operationally as well as being damaging, annoying, whatever. So for both those reasons you would very much prefer to release only males. In A. aegypti we have physical sex-separation methods that work really well. It turns out that the female pupae are quite a bit bigger than the male pupae and you just effectively sieve them. Most insects you can’t do that. Medfly and even Anopheles mosquitoes don’t really have that difference and then you need to go to genetics-based sex separation method.
4. In all places that you think about Dengue as a problem, it’s actually an invasive species. It’s native to a part of Africa and it’s been inadvertently spread around the world by humans relatively recently in the last few centuries, sometimes in the last few decades so from an environmental perspective it’s not a native species in the first place in these areas and there’s no initial reason to think – although you’d want to look on a case-by-case basis – that there would be members of the ecology that would be dependent on them or that it would be a key part of the ecology. So there’s really a whole series of reasons around why we started with this mosquito. You want to start with something that looks like a good target.
5. The mosquito is very robust and easy to rear.
6. Even if you felt there were currently adequate control solutions for Dengue, which I don’t think very many people in the field would say or for malaria, then you should still be developing new ones because the current one have a lifespan that is probably measured in years rather than decades because they will lose efficacy because of resistance.

HN: How would an insect be able to develop resistance to genetically engineered sterility?

LA: There are good reasons to think that resistance will be 1) less of an issue and 2) manageable. I would be very wary of anyone who says their particular intervention has no possibility of resistance. If we could find breeding sites for these mosquitoes and pour concrete in them, they will develop resistance to that. You might say that’s ridiculous but they will because they will start being selected to use a wider range of breeding sites. They will start to use breeding sites that perhaps they wouldn’t have used before because of the lack of current ones. So you could have behavioural resistance to concrete. It would be very foolish for anyone with drugs, vaccines, genetics – any kind of intervention – to say “you can never have resistance to this,” but the fact is that in the 50 year history of radiation-based SIT there has been very little resistance and the obvious one is behavioural resistance. So if the females can in any way recognise and discriminate against the sterile insects versus the wild ones then there would be strong selection for the ones that preferred to mate with wild ones. It has been documented for classical SIT but I’m only aware of one moderately well-documented case in the whole 50 year history of SIT which suggests it’s a pretty hard thing to develop.

You could also imagine biochemical resistance: now something arises in the wild population that neutralises the effect of our lethal genes so although they have it they no longer die because they are somehow resistant to its function. We have looked for that kind of incipient resistance and never seen the slightest hint of it in any of the species in any of the populations that we’ve tested but you have to imagine it could arise and then how do you get around that. If these were chemicals you’d say ‘if you get resistance, what’s your pipeline? Do you have alternatives?’ and we have absolutely heaps of alternatives. Because we’re expressing something inside the cell, there are any number of ways you can kill or disrupt a cell and we can use wildly different biochemical classes of effector against which you would not expect cross-resistance. So firstly we don’t see resistance and secondly we have a wide range of effectors to get around it should it occur.

HN: So how do you breed up a species in huge numbers that carries a lethal gene?

LA: [We use tetracycline-controlled transcriptional activation (TTA)]. In the presence of [the antibiotic] tetracycline you don’t get expression of the effect that’s going to sterilise them or kill them or whatever it is you’re interested in. In the absence of tetracycline [once they are released into the wild] you do. I was very keen to have repressible rather than an inducible sterility because it gives you a biocontainment aspect. These insects are now not wild-type insects. They are fundamentally sterile and you’re keeping them alive by giving them an artificial antidote.

HN: How do you prepare for releasing a genetically engineered strain into the wild?

LA: We have a phased series of trials of increasing scale, first in the lab then in the field. Essentially what you want to do is predict everything it will do in the field short of actually doing it and so that’s a combination of lab experiments and simulation modelling and the two inform each other because the simulation modelling will tell you that some parameters are more important than others or more important than you thought or whatever it is in relation to the field performance. Fundamentally, does the construct do what you want it to do and then have you inadvertently made any changes usually negative performance changes to the strain in the course of the genetic engineering? Do you get the positive effect that you wanted and then have you inadvertently done something to damage the insect along the way? Because if you have you’re back in the radiation situation where you’ve got the sterilization that you wanted but you have also unavoidably damaged the insect in the process. Can you make genetically engineered insects that are reasonably competitive in the wild or are they unavoidably, inevitably weakened in a way that will compromise their ability to find and mate with wild females?

HN: Tell me about the OX513A strain and the journey you have taken to field trials?

LA: [Males are homozygous for a repressible lethal dominant gene that disrupts the development of their offspring so they die as late larvae or pupae]. We developed the strain in about 2002. The key thing is can your males find and mate with wild females? You can test this in the lab but those trials will be in cages of rather small size – 30cm cages. You can say in that – if you put in ten engineered males, ten wild-type females, ten wild-type males – who do females mate? If they are equally competitive on average it would be 50:50. With this strain, we got equal mating. Of course we make very large numbers of constructs and strains and they often don’t perform as well as we’d like so there’s a huge winnowing in the process, as there is in drug discovery. Mating competitiveness, longevity, dispersal. Some of these things you can test up to a point in the lab but it’s very hard to know whether the males can find females over a distance of 10 m or 100 m in a laboratory experiment. So the first experiments were limited releases and the very first thing is just how long do they live and how far do they fly in the wild because we know what a regular wild insect would do, pretty much. And if they are dramatically worse than that it’s a problem. In the lab they live pretty much as long. It’s a classic mark, capture, release experiment. In some of those experiments we released transgenic males with wild males so you can have a direct comparison. How far do the wild-type ones go and how far do the genetic ones go?

HN: The next stage presumably is to find out if they can compete successfully for wild females?

LA: That’s what we did in Cayman in 2009 in collaboration with the Cayman Islands Mosquito Research and Control Unit. We released them relatively uniformly through an area over a four-week period. At the end of that you can look at who the females have mated, which we can do very easily because we just put out artificial breeding sites and the females can lay eggs in them and we collect those eggs, hatch them out and for each one say ‘does it have the fluorescent [DsRed] marker?’ This tells us who the females are mating and then we can also track adults and say what ratio of sterile to wild male we have and just compare those two numbers and that broadly tells us what the relative competitiveness was like.

HN: And how was it?

LA: If you just take the means then in round numbers our males were about 20% of the total males – just under in fact about 16% – and about 10% of the eggs had a transgenic parent – again just under. So you got about half as many transgenics as you’d expect for the male, which is fantastic. [Remember,] it’s a laboratory-adapted strain, reared in the lab, it’s got the transgene in it, it was handled, it was just put out in the field uniformly without any knowledge of where the wild ones were. A whole bunch of those males you’d just put in the wrong place. Net of all of that, to have 50% of wild-type mating competitiveness is fantastic. It’s also way more than you need for success in these kinds of experiments. In big programmes that have worked really well, that number would have been 0.1 or more like 0.01 rather than 0.5. That’s way better than we needed and was extremely encouraging.

That also enables us to say in that area, we released this number and we got this amount of mating so if we want to adjust the amount of mating to half the females mating or something like that how many more males do we have to release to get there? In the next year 2010 that’s what we did. We released the number calculated and with some adjustment got to a level predicted to suppress the target population and indeed it suppressed the target population.

In Brazil we are essentially recapitulating what we did in Cayman. It seems pretty likely that what happened in Cayman will happen in Brazil but it’s a different place, the mosquito population could be different so it’s worth repeating that. It will show whether the Brazilian population behaves the same or whether it doesn’t.

HN: You have another strain in development OX3604C?

LA: The beauty of it is you can get the sex-separation and the sterilization equivalent from the same genetic thing. [Females are flightless, so unable to feed or find males, they die without mating]. With the female specific system you can just release eggs, knowing that they are going to grow up in the absence of tetracycline and only males will make it. If you were going to say we’ve got these isolated communities and you want to send them some material they can use themselves in a totally community-based method really you want to be sending them the eggs. The eggs are fantastic. The eggs will stay dormant, dry for months. They are incredibly light and easy to ship around. 1 million eggs will weigh a gram or something, just nothing. Doesn’t need refrigeration, doesn’t need anything special, just sit around for a couple of months, delayed transport doesn’t matter.
These mosquitoes grow in a very wide range of rather simple breeding sites, which is one of the reasons why they’re such a pest and so hard to deal with. But we can turn that to our advantage by saying well actually they are really very easy to grow. Just add water and you get instant mosquitoes.

HN: How did you come across this strain?

LA: We went out and looked for it. We wanted something that was late, so in pupae, and female specific so we looked at the differences in genes expressed in female pupae and the genes expressed in male pupae. We got a number of them and we analysed them and another group led by Tony James did the same experiment and got some of the same genes. It turns out to be specific to the female flight muscle. It is in fact a flight muscle actin. The indirect flight muscles, which power flight in insects, are very specialised muscles. Drosophila has its own indirect flight muscle actin different from normal flight muscle actin. But it’s the same gene in males and females. In mosquitoes it isn’t.

HN: Why would female have different flight muscles?

LA: We don’t know.

HN: But you must have thought about it?

LA: I have. For that to be maintained, to be advantageous you’d have to think there is some difference in flight between males and females and it’s a little hard to see what that might be. The female has to fly after taking a blood meal so she has taken on a small multiple of her own body weight in blood so has to be able to fly with a much heavier load than a male ever has to. The other is around courtship. The males and females modulate their wing-beat frequency in different ways.
There’s clearly scope for investigating that further. I would love to do that. It’s just not part of my current mission to do that. But you find these things along the way that raise lots of pure biology questions that I would love someone to pick up.

HN: What are your plans for this strain?

LA: We’re planning to recapitulate trials much like what we did with OX513A. I think we will have trials of within the next 18 months or so. The regulators will take whatever time it takes for them to be satisfied with it.

HN: There are presumably lots of people who are opposed to this technology, just because it’s GM. How do you respond?

LA: I don’t think there is a big negative reaction to genetic engineering per se and my reason for saying that is look at genetically modified insulin look at genetically modified vaccines. People inject themselves frequently with recombinant insulin, which is clearly a genetic engineering product, so it’s quite hard to say there’s a fundamental opposition to the technology itself. Ditto vaccines. But clearly there has been in some countries in the context of food. I think part of the reason [for some opposition to GM] is that we’re sitting here in Europe which is one of the more anti-GM areas (though that’s not universally the case even for food) but one of the issues is “where’s the benefit?” In Western countries we’re used to effectively unlimited supplies of clean, cheap, safe food, where is the benefit of applying genetic engineering to the individual consumer, voter, citizen? Maybe there’ll be a few pence off your maize, probably not. Maybe a few less chemicals will be used out in the environment but you don’t see that in the first place and don’t really believe it anyhow. Where’s the benefit? If you can’t see any benefit, why would you agree to some changes, some technology that other people say is scary and dangerous and you’re not too sure about?

Whereas, if you go to a Dengue-endemic country and talk to people about Dengue, everyone knows about Dengue. Everyone has either themselves or a close family member suffered from the disease, it’s a very painful disease, very worrying, there’s no specific therapy for it, there’s no protection for it. Everybody agrees that Dengue is a bad thing and also that current methods are inadequate for controlling it. It’s very well known, partly because of its prevalence, partly because one of the lynchpins of such control as there is is trying to get citizens to clean up breeding sites, to clean up water sources and so on. There’s a lot of public information about Dengue and its association with mosquitoes. Everybody agrees that new measures and doing something about it would be a good thing. So we then have that huge common ground. Now we’re just discussing the method. With GM crops in Western Europe, you just don’t have that common ground. We’re just not in that situation. There are a whole host of other differences with food about the fitness consequences of the transgene and implications for spread. You’re trying to make a beneficial thing stronger by giving things that’ll help it like herbicide resistance and insect resistance and those are traits which you can imagine under some circumstances might possibly spread. Whereas we’re putting in things with huge, huge fitness penalties like sterility and death into something that’s undesirable in the first place and trying to get it to go away and it’s clear that those transgenes are not going to spread even were they to go outside the population area where you want them. Many of these crop plants have weedy relatives with which they could hybridize. These insects have cleaner species so that route for spread is not available. There’s no equivalent of conventional agriculture. So one of the issues with GM agriculture in Europe is its coexistence with conventional agriculture and how are you going to do that, how are you going to separate them, how are you going to avoid genetic contamination between them? You know, there is no established set of conventional mosquito breeders who are going to be upset with our release of geneticially engineered mosquitoes. There is a big health benefit that everyone understands.

We’ve had a little more concern about the GM aspect in the Malaysian press than in Cayman and Brazil. It’s going to vary from one country to another, one culture to another. GM maize in Mexico was such a big issue. GM cotton was much less controversial. It’s going to differ a bit from one country to another but on the whole I think the fact that it’s for health and the big public benefit and public good derived from success really puts us in a different space from GM crops, at least GM crops in Western Europe.

One of the features of this technology is that it protects an area and it protects everybody in that area irrespective of their wealth, power, status or education or whatever, which from an equity basis is quite attractive actually. Our aim is to reduce the burden of Dengue in vulnerable populations but if this helps promote a more nuanced discourse about genetic technology in general, that would certainly be a welcome side effect.

HN: Could this sort of technology be used to control a mammal like the rat?

LA: A large number of adult male rats – sterile or not – is probably not the way you want to go.