Figure 8.

Knockdown of Insr in fifth instar larvae via dsRNA injection delayed pupation time, produced small pupae, and decreased the 20E titer.A, Western blotting showing the efficacy of Insr knockdown. The larvae epidermis protein was extracted after the last injection 24 h; 12.5% gel. ImageJ software was used to transform the image data. B, insect phenotypes after three times injection of dsInsr or dsGFP into the larval hemocoel (1.5 μg/larva, in fifth instar 24 h, 48 h, and in sixth instar 6 h). Phenotypes were observed from 90 h to 144 h after the third injection. C, the statistical analysis of different phenotypes based the experimental group and control group. Each group contains 30 larvae with three independent repeats. % was calculated by the number of phenotypes in the 30 samples. Error bars indicate the mean ± SD of three repeats. Student’s t-test was performed to analyze the difference of the two groups of dsGFP and dsInsr. D and E, statistical analysis of body weight and pupation time. F, midgut morphology observed 90 h after the last injection. G, the 20E titer detection in the hemolymph after the last injection of dsRNA for 90 h. H, qRT-PCR detected the mRNA levels of Insr, Pi3k, and Akt after the last injection of dsRNA for 24 h. I, Insr knockdown decreased AKT phosphorylation after the last injection of dsRNA for 24 h. The antibody against p-AKT was used. J, the hemolymph glucose levels detection after the last injection of dsRNA for 24 h, 48 h, 72 h. The statistical analyses were conducted using Student’s t-test (∗p < 0.05; ∗∗p < 0.01) based triplicate. Error bars indicate the mean ± SD.