1.     What exactly is the Sterile Insect Technique ("SIT")?

The Sterile Insect Technique (SIT) is an environment-friendly, species-specific method of insect control, which has been described as "birth control for insects" SIT has been used very successfully in agriculture for over 50 years.

SIT works by releasing sterile insects of a target species. The sterile males compete with the wild males for female insects. If a female mates with a sterile male then it will have no offspring, thus reducing the next generation's population. Repeated release of insects can eventually reduce the insect population to very low levels or zero and hence reduce the damage or spread of disease.

SIT has been used very successfully in agriculture for over 50 years but is currently restricted by the need to irradiate the insects to sterilize them.  For some species, for example mosquitoes, the dose required to sterilize the males also damages their fitness to the extent that SIT cannot be used effectively.

2.     What is the SIT technology that might be tested in Key West?

The SIT technology being evaluated for use in Key West was developed by Oxitec using genetics rather than radiation. The gene that has been introduced into the mosquito inhibits the cell's ability to function normally. This disrupts cell growth and means that all the progeny die before reaching adulthood making the male effectively 'sterile'.  When a 'sterile' male mates with a wild (non sterile) female all the progeny will inherit this gene and none will survive. This approach is not toxic but it stops the larvae from developing so no adult mosquito emerges.

This means that for the first time the Sterile Insect Technique, that has been extremely successful in agriculture, can be applied to mosquitoes that spread dengue, chikungunya and yellow fever.

3.     What is dengue?

Dengue fever is a severe, flu-like illness that affects infants, young children and adults. There is neither specific medicine nor vaccine for dengue fever.

Dengue fever is transmitted by the bite of an Aedes aegypti mosquito infected with any one of the four versions of the dengue virus. Symptoms appear in 3-14 days (average 4-7 days) after the infective bite. Dengue hemorrhagic fever is a potentially lethal complication, particularly in children, and early clinical diagnosis and careful clinical management by experienced physicians and nurses is necessary to reduce the number of fatalities.

The Aedes aegypti mosquito has spread itself around the world, largely in the last 50 years and, as it has done so, dengue fever has increased dramatically.

More than 70% of the global disease burden is in South-East Asia, Asia and the Western Pacific area. In Latin America and the Caribbean, the incidence and severity of disease are increasing rapidly. Because dengue fever is rapidly increasing and current control methods and tools are not adequate to stop this growth, new approaches are needed.

4.     Do all mosquitoes spread Dengue?

No. The Aedes aegypti female mosquito spreads dengue.  Males do not bite or spread disease (in fact males cannot bite!).

Aedes aegypti is the main vector  to spread dengue,another species Aedes albopictus can also spread dengue but is much less successful as a disease vector. Other mosquito species do bite humans but do not spread dengue.

5.     Is Oxitec's mosquito genetically modified?

Yes.

6.     What is a GMO?

A genetically modified organism (GMO) is an organism whose genetic [material] has been altered using techniques in genetics generally known as recombinant DNA technology.

7.     Is genetic modification bad or dangerous?

No; genetic modification (or recombinant DNA technology as it is sometimes referred) is a scientific tool or technique. GM approaches are regularly used, for example, in the production of pharmaceutical drugs or vaccines, new medical approaches (gene therapy) in industry (production of enzymes) and in new biofuel approaches.

It should be noted that all GMO's must go through extensive scientific testing and a thorough and appropriate Regulatory process before being authorised for use.

8.     What are the benefits of using Oxitec mosquitoes over other control methods?

There are several benefits:

• This approach has the potential to reduce the Aedes aegypti population to a level below which disease is not transmitted. The Aedes aegypti mosquito is well adapted to live with humans in the urban environment; it bites in the day and lays eggs in a wide variety of breeding sites. Aedes is particularly difficult and costly to control using conventional methods such as insecticides because of the difficulty of finding and treating both adults and larval breeding sites.
• It is a method of biological control and is likely to be less costly and more effective than using humans with chemical sprays or fogging devices to kill the biting females. The technique harnesses the natural instinct of the male mosquito to actively seek female mosquitoes of its own species.
• It is an environmentally friendly approach (no toxic chemicals are used) the sterile mosquitoes released will only mate with their own species and hence no other species is affected (unlike, say, where a general insecticide is used).
• The Oxitec mosquitoes that are released are males and hence do not bite or spread disease.
• It also reduces the threat of chikungunya and yellow fever which are also spread by Aedes aegypti mosquitoes

9.     What are the likely impacts on the environment and on humans?

The main impact on human health will be to reduce the number of Aedes aegypti mosquitoes that can spread dengue.

There is no permanent change to the wild mosquito population and therefore unlikely to have any impact on the environment compared to the currently used alternatives.

Aedes aegypti is not originally native to Key West, though it has been present on and off in the last few decades.  It is not a keystone species, there are no birds, fish or other insects that feed exclusively on it and therefore reducing the number of Aedes aegypti is most unlikely to have any impact on the environment. Additionally, the released mosquitoes will die in the environment and their progeny will die so this is a 'self limiting' approach. i.e. there is no permanent change to the wild mosquito population.

• The main impact will be to reduce the number of Aedes aegypti females that bite and spread disease.
• There is no threat to humans as the mosquitoes that are released are males and males do not bite or spread disease.

10.What is actually killing the mosquito larvae/ what genetic modification has been made?

The gene that has been introduced into the mosquito inhibits the cell's ability to function normally.

This means that all the progeny die at the larval stage making the male 'sterile'.  When a 'sterile' male mates with a wild (non-sterile) female all the progeny will inherit this gene and none will survive. This approach is not toxic but it stops the larvae from developing so no adult mosquito emerges. It works only by inheritance and does not affect other insects or predators such as fish or birds that might eat a mosquito.

11.Once the sterilised mosquitoes have mated with the wild female Aedes aegypti mosquitoes, what   happens to them? Do they just die off?

Yes. Aedes aegypti is a relatively short-living species.  Adult males can live up to around 10 days in the wild.  In the laboratory, under ideal conditions, they can live a bit longer, even in extreme cases only up to a month.

12.When will open field release trials be conducted in Key West?  What regulatory approvals are required?

Open field release can and will only take place once all necessary regulatory and ethical approvals have been obtained, based on the results of scientific review. USDA APHIS Veterinary Services are the co-ordinating agency for this trial application.

13.What tests have been done to prove that this technology works and is no threat to the environment?

Oxitec and independent collaborators around the World have conducted extensive testing to make sure that the mosquitoes are only different from the wild ones in their ability to reproduce.

The mosquitoes have also been tested by laboratories around the world including the Institute Pasteur in Paris, the Institute for Medical Research in Malaysia, University of Colorado, USA and UHUHS, USA.  The Center for Medical and Veterinary Entomology at Gainesville have also tested these mosquitoes in outdoor cage trials. The USDA APHIS CPHST has also tested the same approach in other insect species and a thorough examination of the risks and benefits has been conducted under the National Environmental Protection Act (NEPA), which concluded that the use of this technology was environmental preferable.  The same technology, that was reviewed under NEPA and is in the mosquitoes proposed for this trial.   However, laboratory tests will only give information on the behavior of the mosquitoes in an artificial situation.

Oxitec and its collaborators have performed open field release tests of Aedes aegypti in several countries (including the Cayman Islands, Malaysia and Brazil). These studies have shown that our technology can reduce a field population of Aedes aegypti by up to 80%. In addition, when releases were finished no evidence of the RIDL insects in the wild (through genetic marker identification) was seen after 2 weeks (monitored for several months after the trial).

14.Have similar experiments been carried out before?

Oxitec and its collaborators have performed open field release tests of Aedes aegypti in several countries (including the Cayman Islands, Malaysia and Brazil).

There are several related experiments that have occurred in the past.  One trial using sterile mosquitoes was conducted in El Salvador in the 1970s, where 4.4 million sterile mosquitoes were released in a 15 square km area over 22 weeks. (This used a form of chemical sterilisation which due to toxicity, would probably not be permitted for general use today)

This successfully eliminated the target mosquito population.  They then went on to a much larger area, and were able to suppress the population but not to totally eliminate it. Immigration of local mosquitoes into the trial area was the cause.  The need to avoid such immigration, especially for a small trial experiment, is the primary reason for needing a reasonably isolated trial site.

The Cayman Government completed a small scale feasibility study with the same genetically modified Aedes aegypti in 2009 which addressed how long the mosquitoes lived in the environment, how far they flew and whether they would mate with Cayman female mosquitoes.  Approx 19,000 sterile male mosquitoes were released over 10ha for a 4 week period.

A larger experiment, releasing over 3 million male RIDL mosquitoes over a6 month period was conducted in Grand Cayman in 2010 and an 80% reduction in Aedes aegypti achieved in a small area of 16 Ha (~40 acres). The same mosquitoes have also been tested in small scale releases in Malaysia and a large scale trial in Brazil. In addition, when releases were finished no evidence of the RIDL insects in the wild (through genetic marker identification) was seen after 2 weeks (monitored for several months after the trial).

Another example is the Pink Bollworm (Pectinophora gossypiella), a moth that is a pest of cotton.  The US Department of Agriculture has evaluated Oxitec's GM pink bollworms in three years of open-field and mass-rearing trials, and so far have released over 15 million GM moths. Oxitec's strain has performed exactly as predicted in these trials. There were no negative outcomes of any type (whether environmental, agricultural or to human health) detected.  These trials were also subject to rigorous evaluation by the US regulatory authorities before any release occurred.

15.Are there international guidelines or regulations for field trials of GM mosquitoes?

Regulations for the release of GM organisms of any kind in a country are covered by the national (Biosafety) regulations and law of that country. The Cartagena Protocol on Biosafety is an international treaty governing movement of GMO's between different countries.

The Cartagena Protocol on Biosafety is an international treaty governing the movements of living modified organisms (LMOs) resulting from modern biotechnology from one country to another. It was adopted on 29 January 2000 as a supplementary agreement to the Convention on Biological Diversity and entered into force on 11 September 2003.  A sub-group within the Ad Hoc Technical Group on Risk Assessment and Risk Management has prepared guidance on the risk assessment and management of modified mosquitoes.

Regulations for the release of GM organisms of any kind in a country are covered by the national (Biosafety) regulations and law of that country.

Additionally there is now a growing amount of guidance, and training, from the WHO, United National Development Programme (amongst others) for GM insects. For example, WHO-TDR is sponsoring a consortium of experts, MosqGuide of which Oxitec is a member, to develop guidance on the biosafety, regulatory, ethical social and cultural aspects regarding the testing and deployment of genetic vector control methods in disease endemic countries. www.mosqguide.org.uk

16.Have any risk assessments been carried out for this technique?

Yes, risk assessments are routinely carried out by Regulatory Authorities according to their own requirements. Bodies such as the WHO can, and do, provide guidance and training. Every country that evaluates the approach will carry out its own risk assessment.

One such assessment - the Environment Impact Statement has been carried out by the USDA in the on the use of equivalent technology in some agricultural pests (fruit flies and pink bollworm) and concluded (Record of Decision, May 2009) 'that the use of such technology was not merely acceptable, but in fact was 'the environmentally preferred alternative' (http://www.aphis.usda.gov/plant_health/ea/geneng.shtml)

In Malaysia, where contained field trials of this technology have taken place,   the government and UNDP sponsored a workshop on Risk Assessment of Transgenic insects The results of the workshop where a risk assessment was conducted on Aedes aegypti with a self-limiting trait, has been published in the Asian Pacific Journal of Molecular Biology and Biotechnology shortly (Beech et al, 200917(3)) and recently updated by Patil et al (2010) in the same journal (volume 18(2).  The Government of Malaysia has also published it's own risk assessment regarding the trial and it is available on the Biosafety Clearing House website.

In Cayman, the Cayman Islands government have reviewed the risks associated with such open field trials and determined them to be low risk. The trials in 2009 and 2010 released millions of RIDL insects and found there to be no impact of release other than on the mosquito population. In addition, when releases were finished no evidence of the RIDL insects in the wild (through genetic marker identification) was seen after 2 weeks (monitored for several months after the trial).

17.What would happen if some females were released together with the male sterile mosquitoes?

The consequences of a small number of female mosquitoes being released are minimal. Great care is taken when sorting the male pupae from the female pupae in the laboratory. Sorting is carried out in stages with several checking steps to minimise the possibility of females being included.  It is possible to sort the male pupae from the females with high efficiency as the male pupae are smaller than the female pupae.

If small numbers of females are released it is likely that they will die relatively quickly compared to WT females. We have shown that our males that are released do not live as long as wild type males because they are breed in the laboratory.  The females carry the RIDL gene and even if one of these females mate with another mosquito (whether a sterile male or a wild fertile one) then all the progeny will die as larvae/pupae. The females are 'sterile' as well.

However if one were bitten by a female sterile mosquito it would be exactly the same as a bite from a wild one. The released mosquitoes are clean of disease. No DNA is transferred in a bite.

18.How is Oxitec's mosquito different from other genetically modified mosquitoes such as refractory mosquitoes?

Oxitec's approach is unique and different in concept.  Oxitec's genetically sterile male Aedes aegypti mosquitoes will mate with the wild (non sterile) Aedes aegypti females. The offspring that result from this mating of sterile males and wild females will not survive. Therefore the approach is 'self limiting' i.e. the gene is not passed from one generation to another and no permanent change is made to the mosquito population. There are a limited number of genetic modification approaches being researched that aim to stop mosquitoes from transmitting dengue, malaria and other diseases.  These methods require the genetically-modified (GM) mosquitoes to establish stable, permanent, breeding population in the wild that will progressively replace the existing wild population. No such strains have actually been released and all such approaches are at the research stage.

19.Can the introduced genes be transferred to other species?

No, Aedes aegypti mosquitoes cannot breed with other insects in the wild, not even with other species of mosquito. Therefore the genes are restricted to this single species of mosquito

Animals that eat the sterile Aedes aegypti mosquito will be exposed to nutritional elements; protein, fat, sugar and others, as they would from eating any mosquito, but they cannot take up genes through this route.

20.Who is Oxitec?

Oxitec Limited was founded in 2002 to develop and commercialize leading-edged science and biotechnology invented at the University of Oxford in the United Kingdom.

Their approach to insect control was recognised by the Bill and Melinda Gates Foundation Grand Challenges for Global Health Initiative funded by the award as part of a US$20 million consortium developing genetic strategies to control disease-carrying mosquitoes. This award was made following intense scrutiny and evaluation. Oxitec has also received grants from the UK Government to facilitate research and development, as well as the Wellcome Trust, and others. It is working with the World Health Organisation (WHO) on a project to develop best practices in the deployment of genetic control methods against mosquito disease vectors in disease endemic countries.

21.What community engagement has been conducted for field release in Key West?

We have a community engagement plan in place to inform all the relevant people and to have information available on request about the trial. Community engagement activities so far have consisted of;

·         Radio interviews

• Public information events
• Consultation with senators
• Local officials
• Web site information