Genetic control of mosquitoes HEALTHY PEOPLE HEALTHY ENVIRONMENT Genetic control of mosquitoes Luke Alphey, Founder and Chief Scientific Officer Visiting Professor in Zoology, University of Oxford

Injecting DNA into mosquito eggs Oxitec Ltd Based in Oxford, UK, 30 employees Founded in 2002 to commercialise new technology from Oxford University Control of insects Agricultural pests Public Health (mosquitoes) Injecting DNA into mosquito eggs

Introduction Why? How? When? Pest insects cause $$bn damage and transmit major diseases How? Engineered sterile males RIDL®: Release of Insects carrying a Dominant Lethal genetic system Genetics, molecular biology When? Initial strains successfully tested in field Marker-only moth: USA 2006 RIDL mosquitoes: Cayman Islands 2009, Malaysia 2010, Brazil 2011

Increased Risk of Vector Borne Disease Dengue - growing global pandemic Over 100 million cases annually Severity increasing No specific medication or vaccine Chikungunya - emerging threat 1.8 million cases in last 6 years (WHO) Reunion (2005) had 266,000 cases and 254 deaths Italy (2007) – 197 cases and I death Oxitec/Nimmo 4

WHO Dengue control

Male mosquitoes! In fact the sound I played you at the beginning was the love song of two courting mosquitoes. Males are highly evolved to find females. And when they do, in the normal way of things, you get lots of baby mosquitoes, and so the population continues and expands. [But if we can release engineered sterile males into the population, they will court and mate wild females, but have no viable progeny.]

But if we can release engineered sterile males into the population, they will court and mate wild females, but have no viable progeny. We call this system RIDL, so these are RIDL males. If we can release enough RIDL insects for long enough, the target population will decline and collapse. This might sound crazy, but it has some big advantages. The sterile males will mate only with females of the same species, and not with anything else, so there are no off-target effects on other insects. And these males will seek out those females for you. People often ask: can you rear enough RIDL males – after all there are a lot of mosquitoes out there. RIDL®

But if we can release engineered sterile males into the population, they will court and mate wild females, but have no viable progeny. We call this system RIDL, so these are RIDL males. If we can release enough RIDL insects for long enough, the target population will decline and collapse. This might sound crazy, but it has some big advantages. The sterile males will mate only with females of the same species, and not with anything else, so there are no off-target effects on other insects. And these males will seek out those females for you. People often ask: can you rear enough RIDL males – after all there are a lot of mosquitoes out there. RIDL

A Genetic Solution Release engineered sterile males to prevent mosquito reproduction and so control dengue “birth control for mosquitoes” Sterile male mosquitoes actively seek females Find mosquitoes better than human inspectors Based on Sterile Insect Technique How it works Rear millions of insects Sterilise with irradiation Release over wide area Sterile males mate with wild females: progeny don’t survive Pest population declines Species-specific Used for over 50 years WHO

New World Screwworm Eradicated New World screwworm eliminated on a continental scale by a rolling programme of release of sterile insects. 10

RIDL: fail-safe / replacing radiation RIDL insects are genetically sterile Repressible Release homozygous males X Bi-sex lethal Female-specific lethal X Introgression of genes through male line

RIDL®: molecular biology Controllable Gene Expression Antidote (Tc) Female death death tTA effector promoter tetO Female specificity Thomas et al. 2000 Science 287: 2474-6 Fu et al. 2010 PNAS 107: 4550-4

Act4-tTA + tetO-DsRed (DsRed) tTA effector OX3545F Act4 promoter tetO + promoter OX3545F Flight muscles only

OX3604C RIDL mosquitoes Males Females Flightless mosquitoes cannot survive in wild (or find hosts). Unable to mate even in laboratory. Males have normal flight ability, as have females given antidote as larvae.

fsRIDL phased trials – large lab cage trials RIDL cage trials performed in Colorado State University (Megan Wise, Bill Black) showing suppression of target population

RIDL: fail-safe / replacing radiation RIDL insects are genetically sterile Repressible Release homozygous males X Bi-sex lethal Female-specific lethal X Introgression of genes through male line

Development trials First open release Grand Cayman 2009 10km First open release Grand Cayman 2009 mating of RIDL males to local females excellent mating competitiveness provided data for suppression trial 2010 500m Each Area approx 16 Ha (40 acres) No conventional control for Aedes aegypti Release period May-Oct 2010 with pre- and post-release monitoring

Cayman field trial 2010 Trial was complete success; all endpoints met Cayman field trial 2010 Aedes aegypti populations show seasonal variation in Cayman, driven by rainfall. Control area – Area C, green line – shows this pattern (as did other control areas, omitted for clarity), illustrated by fitted polynomial trend line. Release area – Area A – shows clear suppression from beginning of August. Release period finished Oct 15. for last 7 weeks, release area had average ovitrap index of 10% vs control area 50% - an 80% reduction in the treated area relative to control. Since ovitrap index is somewhat non-linear, this probably represents suppression of the mosquito population by somewhat more than 80%. The degree of suppression is limited by immigration from adjacent areas, e.g. Area B, which was untreated and had a high mosquito population (resembling Area A). Note that the control and treatment sites are close, so share environmental effects. The two plots therefore largely parallel each other in terms of week-to-week variation, with the additional effect of suppression in Area A. Trial was complete success; all endpoints met Clear suppression from early August Sustained release of RIDL OX513A males can suppress a field population of Aedes aegypti mosquitoes Maximum degree of suppression limited by immigration GM mosquitoes can perform successfully in the field

Bringing new technology to the field Technical Community Regulatory RIDL Nature

Regulatory Progress The USDA completed (2008) an Environmental Impact Statement (EIS) on the use of autocidal technology (RIDL) in fruit flies and PBW Record of Decision: this is the environmentally preferred alternative North American Plant Protection Organization (NAPPO) standard signed late 2007 MosqGuide: WHO/TDR develop guidance for use of GM mosquitoes for disease control Oxitec transgenic insect approvals Multiple movement and contained trial approvals (Medfly, Mexfly, pink bollworm, Aedes aegypti, Ae. albopictus) Open field release approvals in the USA (PBW 2006,7,8) Released 15 million Oxitec pink bollworm from aircraft over 2500 acres (2008)

Community Engagement Focus Group: reactions to Oxitec approach breakthrough! good news kills larvae good – something worth trying going forward provides immediate solution / result in preventing Dengue seen as a long term solution cf other methods which are all seen as short term some even welcome the research team to test the technique in their community From TNS Malaysia Community engagement Malaysian Health Minister Datuk Seri Liow Tiong Lai “We see it as the most efficient and fastest way in eradicating Aedes mosquitoes from our local environment,” Liow said, adding that Aedes is not a species endemic [native] to Malaysia. Monday 11th Oct 2010

Frequently Asked Questions Will the genetic modification spread outside the release area? The released mosquitoes and their progeny will die so this is a ‘self limiting’ approach, with no permanent change to the wild mosquito population. The large fitness cost (and no component advantage) also prevents spread. Do mosquitoes provide valuable ecosystem services (e.g. food chain, pollinators)? Aedes aegypti originated in Africa and only achieved pan-tropical distribution in the 1930s. Therefore in most countries it is not a native 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 negative impacts on the environment. If one mosquito suppressed, will a worse one replace (niche replacement)? Aedes aegypti it occupies an unusual, human-associated niche normally empty in its absence. Aedes albopictus, a potential alternative, is an inferior vector of dengue. RIDL strains are also available for Aedes albopictus. Can we release enough (feasibility, economics)? Long history of success and data from SIT implies ‘yes’, as do modelling and data so far.

HEALTHY PEOPLE HEALTHY ENVIRONMENT Acknowledgements Greg Simmons Bob Staten (rtd) Tom Miller (UCR) Angi Harris Bill Petrie IMR, Malaysia Lee Han Lim PI: Tony James PI: John Mumford 23

Thank you… 24