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Control zest tha rane Anopheles gambiae mesa a “deplayng characters white ndages elongated pap Univers iting MOLECULAR APPROACHES TO ALTER OLFACTORY-DRIVEN BEHAVIORS OF INSECT VECTORS Richard Axel, Columbia University, United States Leslie Vosshall, The Rockefeller University, United States Leslie Voshalloined Nobel Prize-winner Richard Axels neurobiology laboratory at Columbia University as young scientist, Her charge: Idenily how the fruit ly Drosophila melanogaster employs sophisticated sense of emell to direct its behavior "Twenty years later, through a series ‘of wists and turn, that initial scientific journey plumbing the biology of odor detection hat turned Vosshal into one of the leading authorities on why. mosquitoes bunt humans, Discoveries she iret made during seven years of single-minded receatch in Axel’ lab have Jed toa lab of her own at The Rockefeller ‘University a prestigious appointment aca Howard Hughes Medical Investigator, and invitations to speak around the ‘world. And, withthe help ofa five-year, 4$5 mailion grant from the Bill & ‘Melinda Gates Foundation’ Grand ‘Challenges in Global Health program, Vosshall, in collaboration with Axel, has “uncovered a new way to prevent malaria, by jamming mosquitoe® ability to stale and bite humans Finding new ways to shield humans from the mosquitoes that catry malaria ie ‘vital front in the global bate agninet the disease. Each year about 600,000 peeple ein Africa and other tropical ‘most of them children —de to bite ftom the Anopheles gambiae mosquito that ‘ates the malaria parasite Plasmodivm: {Jaleiparum. The female Anopheles wssaly bites inthe late evening to abtain blood swith the protein-rich nourishment that her fertilized eggs nced to mature. “Thats why bed-nets coated with pesticides have gained favor as an effective way to counter the deadly infections. As the Ansects have developed resistance tothe pesticides, developing new ways to thwart the bugs has become a major objective of the Gates Foundation and other public health organizations. In the early 1990, Axel and Voeshall (pronounced Voss-hall) were using new laboratory tools to explore the biology ofsmell The bate agsinet malaria was not part oftheir plan, "Neither Leslie nor [had ‘ny knowledge of insect-borne diseases ‘most common in the developing world” ‘Axel recalls, And yet the central finding oftheir collaboration, a gene called oreo fet characterized by Vosshall in fruit fies, prompted the two scientist to begin mapping the neurobiclogy tht i vital to the spread of insect-borne diseases. "We believe the best way to interfere with insect behavior lke biting humans {sto first identify the many elements of that behavior atthe very basic molecular and cellular level” Axel says “Any othe approach just worst work” Pareuing the orco gene met that criterion, But unraveling the gene’ critical role in the mosquitoe® pursuit of human blood involved years of perseverance and frustrating setbacks. Tn the end, the researchers discovered that orc produces a protein in the nerve cells of the mosquitos antennae, which i esental forthe insect to sense most odors—sich a the distinctive scents humans secrete in their oweat and breath, When Vosshll realized thatthe (Oreo protein is a central olfactory switch common to all insects, the gene—andl the protein it generates—became a prime tage for disrupting all kinds of bug behaviors, That discovery has led to new pptoaches in pesticide esearch, including sing molecular genetics to igure out exactly how insects detect humans in order to develop compounds that keep {hem away from ue. Several companies, including Bayer CropSciences of Germany, ate seeking broad-spectrum agricultural pesticides that selectively deactivate the Oreo protein ‘The company initiated its work on Oreo asa result of collaboration with Axel and ‘Vosshall that was funded with $350,000 lover two years, part of a $2 million two- {year extension gran from the Gates Grand Challenges in Global Health program ‘While Bayer is continuing to work on Oreo inhibiting repellant fr agrcultral Pests, the company says it ended is effort lo develop a mosquito-specific product ‘when the grant concluded in 2012, ‘When Vosshall joined Axel’ ab back in 1993, ithad been just two yeas since ‘Axel and Linda Buck! had identified the nerve receptors that rodents we to detect ‘odors-a discovery for which they were awarded the Nobel Prize in 2004, Since ‘he Drosophila fy has long served as a model for investigating cellar procestes in higher forms of life, including humans, "We thought would be an important achievement to find Axel-Buck receptors in the fly, and that finding them would be prety straightforward” Vosshall ays "We were wrong” ‘After four years of “lure afer fre” ‘vith Vosshall and fellow post-doctoral researcher Hubert Amtein sifting bit by bit through the DNA in the flys antennae for genes similar tothe ones found by ‘Axel and Buck in tats “we had to concede hat ingocts must wee a totally diffrent ‘mechanism than vertebrates” Vosshall says, "We exhausted every possibility looking forthe obvious: ‘At Axels suggestion, Vosshall went back to bases, testing Drosophila genes for those that met three assumptions ‘olfactory genes in the fy had to code for proteins located in cells in the antennae the proteins chould have azo-clled transmembrane structure” mesning ‘they sit astride a cells outer skin, able to transmit external signals—like odors across the cell membrane, creating an clectrical signal to the brain; and there hhad tobe a large number of closely related ‘genes to accommodate the many aromas released by plants In 1998, wit the genes still unknown, ‘Vosshall’s hunt received an invaluable ‘boost when two separate research groups? produced a preliminary sequence ofthe Drosophila genome (a full genome map ‘was completed in 2000). The team used sofware to scan for DNA sequences that ‘encode for transmembrane proteins and ‘were able to pluck out 10 gene groupe ‘that met their eiteria? Meanwhile, John. Carlson and athers were also using the genome sequence fo identify candidate ‘odorant receptors ‘Even soi ell took another five yeare to identify the fall gene forthe Orco (¢hort for olfactory receptor co-receptor) protein and detail precisely how it works. By that time, Vosthall was running her ov labsts central role” says Vosshall. Her ab also = determined tha each odorant-specific z receptor subunit needed to lock onto the : ‘Oreo protein—much as two Lego pieces lick together—in order to open a neural circuit identifying a scent. Vosshall says she inquired about possible funding from the US, National Institte of Allergy and Infectious Diseases for a grant to further explore the Drosophila olfactory ‘mechanism, but she was turned down because it wasn't deemed valuable to ‘understanding human disease, Work on veetor problems, they told her, must be done on the vector insect, not modeled in ‘ies or moths. ‘About then, Axel says, hisinterest 3 Las Yossal and ahangy eal mosgut te Recker University mosque rom sn public health problems in Arica : ves pigued forthe Sst tme during an i athe Rockefeller University Sheané The other subunits ways Orco, When unseated vist there." started thinking i others had found that oor molecules the odorant specie subuaitand Oxco, about how litle time and effort science passthrough in pores in insects’ the common subunit come together they sn the US. as compated to Europe was Sensory hairs called senila Sensila, create signaling pathway that lows the focused on health problems i the like tiny noses, populate insect antenmac insect to identi a specific odor developing werd” he sys. Thats one and another appendage ona bugs head Ina groundoveaking study published in reason the requet for proposals in 2008 called masillay pulps rom there, the 2004, Vossalland her lab at Rockefeller from the newly created Grand Challenges ‘odor molecules low onto nerve cells that reported that es engineered to contain in Global Heath aught his attention. ‘contain odorant receptors (ORS) elective versions of Orco were unable “Tewas immediately apparent to me that ‘Te biggest surprise cme fom to rexpond to diferent sents even when we could eanlae ou findings in the fly ‘experiments that showed that in oder for genes forthe odarant-speci subunile ta other insects he ay an insect to dently aspecic odor ithad remained itact'Tn addition, nike other "Our easoning was pretty simplistic toatimelatea specific OR thal contains odor-delectng receptors thal varied from thal we could develop compounds two protein subunits One afthe OR _insectto insect, Orco was consistently globally disrupt the insects sensory subunits is sensitive to alimited number of observed among all insects tested system he say. With the grant in hand, ‘odorants. There are many diferent genes “Ttreally took 10 yeats of zeseateh to “Leslie began transitioning her lab from. ‘encoding these odorant-specific subunits, fully soate the oreo gene and determine Drosophila to mosquitoes”THE SCIENCE: ‘Vosshall used the funding to test the more than 100,000 molecules in ‘Rockefellers chemical databace for one that ‘could turn off the Orco proteins switch ina mosquito, "We didn't know what we ‘were looking for so we tried everything; ‘Vosshall says. I took almost tree years just to perfect the test ealed an assay, ‘which required growing a culture of celle that express the orc gene. "Well putin the _gene and come back the next days and the ‘eels would be dead. she says. And when ‘we got the gene in, we coulda get itto ‘express the protein, Richard ab and ous nad to develop all sorts of ophistcated zich to make the system work” In 2010, just when the grant wae ‘renning out, Vosehalls lab found several related molecules that disable the action of the mosquito ORs by acting ‘on the Oreo subunit, But to develop a ‘commercial product—the goal specified by the Gates Grand Challenges in Global Health grant—the researchers needed ‘the help from an industry partner. Axel traveled to Germany and, armed with 4.82 million extension ofthe grant into 2012, he persvaded Bayer to join a three ‘way partnership in which the company’ received $350,000 over twa years to ‘expand on Vosshall work. ‘Within six months, Bayer had screened 4 few million compounds sing Vosehalls cell assay, and by the end ofthe fret year the company’s scientists narrowed ‘the search to several clases of chemicals“The Bayer team found thatthe compounds blinded mosquitoes to humans and similarly dirupted moths and other crop- destroying pests. "Bayer accomplished in six months what took us four yar Vosshall sys ‘Meanwhile, during research conducted ‘under the Grand Challenges in Global Health grant, Vosshall identified — first i fies and later in mosquivoes— the receptors in antennae neurons that respond to carbon dioxide in human breath* Mosquitoes engineered with ‘mutations alfecting the carbon dioxide receptor were slower to rexpond tothe presence of humans? And in other studies, Vosshall hs found that mosquitoes that ‘transmit human diseases uiize both ‘catbon dioxide and odorant receptors to {ind alive human on which to feed “Te urns out mosquitoes have multiple ‘ways to find humans? Vosshall says. “Ourbeliefnow is that a really effective tepellant will have to contain a ‘combination of compounds that diable the Orco protein and CO,” At present, however, no company is pursuing this avenue of research, Commercial interest is likely to emerge when further research shows an Orco targeting compound can lead to an agricultural product as wells a ‘mosquito repellant. Companies have told the researchers that commercial profits are necessary to justly producing a low-cost repellant forthe developing world, \Vorshall is continsing work fet begun under the Gates Grand Challenges in ‘Global Health grant, and is “completely focused on understanding mosquito behavior” she says. Her lab is now studying the biology of how a blood mesl changes female mosquito behavior Fora few days after a blood meal, females lose interest sn humans, So harnessing this natural suppression in behavior may also yield sways to interfere with the spread of malaria Vosshal is using her Hovrard Hughes fanding to support the research andin 2010 also received a $600,000 grant from the [ational Institute of Allergy and Infectious Diseases to help back the labs work “The (Grand Challenges in Global Health) grant has changed my careet” she says. Tm soley focused now on studying mosquitoes and I'm optimistic that our continuing studies will eventually lead to 8 new repellant” * Backat the tine wasa postdoc in Anes lab and ‘snow a he Bred Hulchinson Caner Reach Center Seale here se eaves the mechanisms unerjing the sense af smell and pheromone sensing a mamas * The Berkley Drosophila Genome Projet and ‘the Enropean Drosopila Genome Projet > Voshall 8, Amen H. 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