Figure 3.

Generation of stably-transformed yeast interfering RNA larvicides that induce high rates of larval mortality. (a) Yeast integrating plasmid pRS404/406 constructs for integration of shRNA hairpin expression cassettes placed under control of the Gal1 galactose-inducible promoter were prepared and used to generate stable transformants. #52, #101, and control shRNA expression cassettes were integrated at both the S. cerevisiae URA3 and TRP1 loci. (b) Dry heat-inactivated tablets formed from strains with the #52 hairpin expression cassettes or the #101 hairpin cassettes integrated at both loci generated significant larval mortality in both Liverpool (b) and Trinidad (c) strain larvae, while animals fed with yeast expressing control shRNA lived. The data in b were compiled from two biological replicate experiments, each containing four replicates of 20 larvae, while data in c were compiled from three biological replicate experiments with three replicates of twenty larvae; the data are represented as % Mortality ± SEM. Data were analyzed by ANOVA with Tukey’s multiple comparison test (***P = 0.0001; significant differences exist between #52 vs. control-treated animals and #101 vs. control-treated animals). Dose-response curves depicting the mass of #52 (d) or #101 (e) stable yeast interfering larvicide vs. the percentage of Trinidad larval mortality are shown. LD50 values for #52 and #101 yeast interfering RNA larvicides are indicated. Further details regarding calculation of lethal doses are provided in the methods.