Based on the pattern of AeCPA promoter-based expression, impairing of the RNAi pathway was supposed to last for only 36 h during digestion of the bloodmeal in the midgut. Before the onset of Aa-dcr2 mRNA silencing in midgut cells of Carb/dcr16 females, most likely there were sufficient quantities of dicer2 protein synthesized, which could turn the RNAi mechanism this website against itself. Possibly during the entire 36 h period of RNAi silencing certain quantities

of functional dicer2 prevailed in the midgut cells so that the pathway was compromised in its efficiency and capacity but never completely shut off. Similar lack of complete inhibition of RNAi was observed before when transiently silencing dcr2 in Drosophila S2 cells [27]. This could explain the pattern of find more the Aa-dcr2 mRNA expression profiles in Carb/dcr16 females, where the efficiency of Aa-dcr2 mRNA silencing fluctuated over time but its expression was never eliminated. Moreover, infection with SINV resulted in increased Aa-dcr2 mRNA accumulation in Carb/dcr16 females, showing that the midgut epithelial cells were still able to mobilize additional dicer2 protein, even though the pathway was impaired in the midgut tissue. Increase in Aa-dcr2 mRNA accumulation confirms earlier findings that the TR339 strain of SINV triggers the

RNAi pathway in Ae. aegypti [3]. However, no mechanism for Aa-dcr2 induction has been described so far. We have no clear explanation as to why at 2 days pbm Aa-dcr2 mRNA levels were increased in both HWE and Carb/dcr16 females. We observed that levels of transgenic Aa-dcr2 silencing varied considerably between the different transgenic mosquito lines that were initially tested. This could be caused by corresponding variations in Aa-dcr2 IR RNA expression levels. Based on

previous observations with transgenic mosquitoes expressing a marker gene in midgut tissue (A.W.E. Franz, K.E. Olson, A.A. James, Enzalutamide unpublished results), the TE integration site in the genome of the mosquito can strongly affect gene-of-interest expression levels. Even though maximal silencing of Aa-dcr2 in LCL161 in vivo midguts of SINV-TR339EGFP infected Carb/dcr16 females appeared to be no more than ~50%, it had profound effects on intensity of infection, midgut infection and dissemination rates of the virus at 7 days pbm. Average virus titers in midguts increased from 1750 pfu/ml in HWE to 14,000 pfu/ml in Carb/dcr16 mosquitoes. Accordingly, midgut infection rates increased from 33% (HWE) to 69% (Carb/dcr16) and virus dissemination rates from 30% (HWE) to 60% (Carb/dcr16). These data suggest that the RNAi pathway in the mosquito midgut tightly controls SINV infection by modulating its replication. Thus, MIB and MEB for SINV-TR339EGFP in Ae. aegypti were virus dose-dependent and in this way affected by the RNAi pathway.