Fig 3. Production of ZIKV-derived siRNAs in infected WT and transgenic mosquitoes, and RNA-seq transcriptomic analysis of CpA-Dcr2 and CpA-R2d2 transgenic mosquitoes as compared to WT at 24 h PBM.

(a–c) WT and transgenic (CpA-Dcr2 and CpA-R2d2) mosquitoes were given a ZIKV-infected blood meal, and midgut samples were collected at 2 and 4 dpi for small RNA sequencing. Size distribution and 5′ base frequency of ZIKV-derived small RNA sequences from 3 mosquito groups at 2 time points. (d) Relative expression of total ZIKV RNA, as a measure of viral load, in the control (WT), CpA-Dcr2, and CpA-R2d2 midguts at 2 or 4 dpi. The AeRps7 gene was used as internal control with at least 3 biological replicates, the bar indicates the mean value +/− SEM. (e) The number of DEGs within each FG between the midguts of transgenic and WT. (f) For each FG, the percentage of enriched or depleted transcripts in the midguts of transgenic lines as compared to WT. The total number of significant DEGs in both CpA-Dcr2 and CpA-R2d2 mosquitoes is shown in the Venn diagram. (g) Pathway analysis of both Toll and IMD pathways indicates immune genes in these pathways are strongly up-regulated. The boxes colored in pink represent genes up-regulated in either or both pathways, letters in red or blue or purple indicate genes up-regulated in CpA-Dcr2 or CpA-R2d2, respectively, or in both transgenic lines. (h and i) Log₂-fold change in up-regulation of immune genes in either CpA-Dcr2 (h) or CpA-R2d2 (i) transgenic mosquitoes. Three biological replicates are given for each gene. The bar indicates the mean value +/− SEM. a, P < 0.05; b, P < 0.01; c, P < 0.001; d, P < 0.0001. Detailed information of these up-regulated genes is listed in S3 Table. The illustration was created with BioRender.com. Data underlying this figure can be found in S2 Data. CS, cytoskeletal and structural; CSR, chemosensory reception; DEG, differentially expressed gene; DIV, diverse functions; DIG, digestive; dpi, days post-infection; FG, functional group; IMM, immunity; MET, metabolism; PBM, post-blood meal; PROT, proteolysis; RSM, redox/stress/mitochondrion; RTT, replication/transcription/ translation; TRP, transport; UKN, unknown functions; WT, wild type; ZIKV, Zika virus.