a, Regulation of immune-related genes in the midguts of A. aegypti. The midguts of mosquitoes that acquired 10 µg purified DENV2 sNS1 or BSA via blood meals were dissected for RNA-Seq. Immune-related genes were clustered according to immune pathways and factors. b, Validation of immune-related gene regulation. Expression levels of the immune-related genes listed in Supplementary Fig. 6 were assessed in the mosquito midguts by qPCR at 18 h post DENV2 sNS1 feeding. Gene regulation is represented as the mRNA ratio between sNS1-fed and BSA-fed midguts. The primers are described in Supplementary Table 2. c,d, ROS activities in the midguts of mosquitoes fed DENV2 sNS1: the midguts of mosquitoes fed either DENV2 sNS1 or BSA were dissected for H2O2 detection (c) and dihydroethidium (DHE) staining (d). In d, nuclei were stained blue with To-Pro-3 iodide. Images were examined using a ×10 objective lens on a Zeiss LSM 780 meta confocal microscope. Scale bars, 100 µm. In b and c, values in the graph represent the mean ± s.e.m. A non-parametric Mann–Whitney test was used to determine significant differences. e,f, The role of the ROS system in DENV infection of mosquitoes. Either 3.3 mM vitamin C or 1 mM uracil mixed with 1 × 106 p.f.u. purified DENV2 virions and human blood was used for a mosquito blood meal. Mosquito infectivity was determined by TaqMan qPCR at 8 days post blood meal. g,h, Genetic suppression of the JAK-STAT pathway and the ROS system enhanced DENV2 infection in mosquitoes. GFP dsRNA served as a negative control. Mosquito infectivity was determined by TaqMan qPCR at 8 days post blood meal. In e and g, the number of infected mosquitoes relative to total mosquitoes is shown at the top of each column. Each dot represents a mosquito. In f and h, differences in mosquito infective ratios were compared using Fisher's exact test. P values were adjusted using the Bonferroni correction to account for multiple comparisons. Differences were considered significant if P < 0.017 (f) or P < 0.01 (h). Experiments reported in all panels were biologically repeated at least three times with similar results. NS, not significant.