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Fig. 1 | Parasites & Vectors

Fig. 1

From: Characterization of a broad-based mosquito yeast interfering RNA larvicide with a conserved target site in mosquito semaphorin-1a genes

Fig. 1

Silencing Aae sema1a induces larval mortality. a siRNA #460, which corresponds to sema1a, was identified in a screen [25] in which Ae. aegypti larvae soaked in 0.5 μg/μl siRNA #460 died (compare to control siRNA-treated larvae which survived). Screen data from two replicate experiments (n = 20 larvae/replicate) were combined and assessed with the Fisher’s exact test. shRNAs corresponding to the #460 or control siRNA sequences were expressed in S. cerevisiae from a plasmid (b) or following integration of two copies of the shRNA expression cassettes into the S. cerevisiae genome (c). Larval consumption of inactivated dried yeast interfering RNA tablets corresponding to the #460 target sequence induced significant Ae. aegypti larval mortality (b, c). Data shown in panels b (n = 12 replicate containers/treatment, each with 20 larvae for a total of 240 larvae/treatment) and c (n = 12 replicate containers/treatment, each with 20 larvae for a total of 240 larvae/treatment) were combined from three biological replicate experiments and analyzed with t-tests. For each container replicate in b, c, and all subsequent figures, one 70 mg yeast tablet was fed to 20 larvae unless indicated otherwise. d Ae. aegypti larval consumption of inactivated dried sema.460 tablets prepared from stably-transformed strains (which were used for the assays in this panel and all subsequent trial data reported in this investigation) induced death during the L4 larval or pupal stages (days 4–8), while larvae that consumed control yeast survived to adulthood. e A dose-response curve shows that larval mortality is linearly correlated (R2 = 0.9835) with the dose of sema.460 provided to larvae. Each point on the graph corresponds to the percentage mortality observed in a single container replicate assay performed on 20 larvae (data were compiled from three biological replicate experiments). sema.460 LD50 values with upper and lower confidence limits (CL) are shown. f Larvae that survived treatment with sema.460 were bred, and the susceptibility of their offspring to sema.460 was assessed. No significant differences in larvicide activity were noted in the F0 vs F1 generations (for each generation, n = 6 container replicates/condition, each with 200 larvae that were fed with 10 tablets); data were combined from two biological replicate experiments and analyzed by ANOVA. g When sema.460-treated larvae reared as individuals were fed with one 4 mg tablet, 100% mortality was observed (all control-treated individuals survived). Data on a total of 60 individuals/treatment (combined from three biological replicate experiments) were analyzed by Chi-square analysis. The data presented in panels ac, f and g correspond to mean percentages of mortality, with error bars here and in all subsequent figures representing SEM. ***P < 0.001 with respect to control-treated larvae in all panels

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