Figure 4

Non-infectious bloodmeal depletes Dcr2 transcripts; TSN transcript levels are enriched in mosquito midguts during SINV infection. (A) Effects of bloodfeeding on Ago2, Dcr2, and TSN transcript levels. Relative midgut transcript levels from bloodfed mosquitoes were normalized to unfed controls. Transcript levels at 1.0 indicate no change over unfed control levels. (B) Effects of MRE16-eGFP infection on midgut transcript levels relative to un-infected bloodfed controls. (C) MRE16-eGFP (-) strand RNA detected in midgut total RNA from panel (B). (D) Effects of TR339-eGFP infection on midgut transcript levels relative to un-infected bloodfed controls. (E) TR339-eGFP (-) strand RNA levels in midgut total RNA from panel (D). Mosquitoes were fed either a bloodmeal or the virus indicated. (A, B, D) Total RNA from pools of 5 midguts were used for qRT-PCR of Dcr2, Ago2, TSN and Act1 transcripts. Changes in relative transcript levels of each RNAi component were determined by using actin as an internal reference standard and normalizing each value to that of the control group indicated. Relative expression levels were determined using the comparative Ct method; transcript levels at 1.0 indicate no change over bloodfed control levels. Significant changes in expression levels are shown by asterisks (p ≤ 0.05, Mann-Whitney U test). Error bars depict standard error of three independent feedings. (C, E) Viral transcript copy numbers per nanogram midgut RNA were determined by two-step quantitative RT-PCR; these were calculated using the standard curve method. Error bars indicate standard error of three independent virus infections.