uirement for a gain-of-function mutation to confer DM1 web resistance further suggested that the mutations affected an essential gene, and it is known that both unc-17 and cha-1 function are required for nematode viability. All mutants characterized had sequence changes in the coding region of unc17 that resulted in amino acid substitutions, providing compelling evidence that unc-17 is the locus encoding resistance. The C203Y and Y411N mutations were each recovered in two independent lines, suggesting that very few changes allow for both Spiroindoline resistance and nematode viability. Generation of Resistance to Spiroindolines in Drosophila Melanogaster To confirm that the results from nematodes were applicable to insects, we tested whether the same amino acid substitutions could also generate resistance in D. melanogaster. Wild-type, and variant forms of D. melanogaster VAChT were ectopically “1659636 expressed using the Gal4-UAS modular misexpression system. Expression of the variants was driven by the cha promoter, thus mimicking the endogenous expression pattern of vacht. Multiple independent lines were tested for each variant and viable insects with no remarkable phenotypes were recovered for all. Flies over-expressing the wild-type form of the vacht gene, cha.vacht, were at least 3-fold less susceptible than control genotypes to SYN351-mediated mortality, demonstrating that Spiroindoline Insecticides Act by Inhibiting VAChT simple over-expression of VAChT protein conferred resistance to SYN351. Flies over-expressing one mutant form of the vacht gene, cha.vachtY49N, were completely insensitive to all doses of SYN351 tested over an extended period of time. These results indicated that a single amino acid change in the VAChT protein is capable of generating very high levels of resistance, and that the expression of VAChT in a wild type background is sufficient to overcome the toxic effect of this compound in Drosophila. Thus the mechanism of resistance to SYN351 translates from worms to flies and relates to the function of VAChT. None of the flies engineered to over-express the E341K mutant form of the vacht gene, cha.vachtE341K, were resistant to SYN351-mediated mortality. These flies did not even exhibit the same level of resistance as Cha.vacht flies. One explanation for this observation is that the VAChT E341K variant protein is either not expressed or is unstable relative to the ectopic expression of VAChT, and this inference was supported by the failure to detect elevated levels of the protein by western blotting. A High Affinity Binding Site for Spiroindolines in Insect Tissues and its Relationship to the Vesicular Acetylcholine Transporter It remained a possibility that ” Spiroindolines did not act directly at VAChT, either because mutations in VAChT are able to compensate for other effects, or because changes to the transport activity of VAChT were able to reduce exposure. All genotypes are described in the online methods. Resistance factors for ectopic expression of the wild-type vacht transgene in two 
independent lines, as compared to the parental genotypes ). Resistance factors for ectopic expression of the vachtY49N transgene in three independent lines, as compared to the parental genotypes. Since no mortality was observed at the highest dose tested, resistance factors are the minimum expected based on estimates of LD50 that assume parallel dose response curves to those observed in. Resistance factors for ectopic expression of the vachtE3
