Fig. 3

Double RNAi knockdown of AaCHYMO and AaLT in individual Ae. aegypti mosquitoes. (A) Western blot (WB) analysis showing that the double knockdown led to an effect on AaCHYMO expression (seven individual mosquitoes) and compared to the FLUC control (two individual mosquitoes). α-tubulin was used as an internal control. The WBs were cropped for clarity. (B) Quantification of AaCHYMO protein expression levels in midgut extracts with the RNAi double knockdown of AaCHYMO and AaLT compared to FLUC. (C) Western blot analysis showing that knockdown of AaLT has no effect on AaCHYMO expression. α-tubulin was used as an internal control. The WBs were cropped for clarity. (D) Quantification of AaCHYMO protein expression levels in midgut extracts with the RNAi knockdown of AaLT compared to FLUC. E. Quantification of AaCHYMO mRNA levels in mosquito midgut tissues with the RNAi double knockdown of AaCHYMO and AaLT compared to FLUC. (F) Quantification of AaLT mRNA levels in mosquito midgut tissues with the RNAi double knockdown of AaCHYMO and AaLT compared to FLUC. (G) Quantification of AaLT mRNA levels in mosquito midgut tissues with the RNAi knockdown of AaLT compared to FLUC. (H) Quantification of AaCHYMO mRNA levels in mosquito midgut tissues with the RNAi knockdown of AaLT compared to FLUC. Experiments in (B, D, and E-H) were conducted from 3 biological cohorts using 5 to 12 mosquitoes. Data are presented as mean ± SEM. Statistical significance is represented by stars above each comparison (unpaired Student’s t-test; **** p < 0.0001 compared to the RNAi-FLUC control samples, ns = no significance). (I) Activity assays comparing the RNAi AaCHYMO alone knockdown versus the AaCHYMO/AaLT double knockdown using the MeO-Suc-Arg-Pro-Tyr-AMC substrate. No significant changes in overall activity were observed in the double knockdown (4 to 7 biological cohorts using 3 mosquitoes were tested). Please note that the original blots for (A and C) are presented in Supplementary File 1.