Fig. 2

Reproductive phenotypes associated with RNAi knockdown of Nasrat, Closca, Polehole, and Nudel in Aedes aegypti mosquitoes. A Representative eggs are shown from mosquitoes microinjected with dsRNA against Fluc (control), Nasrat, Closca, Polehole, and Nudel. B The effect of RNAi knockdown on fecundity was studied during the first gonotrophic cycle. Each dot represents the number of eggs oviposited by an individual mosquito. C Melanization of these eggs was examined under a light microscope and a melanization percentage was determined. D An effect of RNAi knockdown on egg viability was examined by hatching eggs 1 week after oviposition. Each bar corresponds to egg viability from 24 to 27 individual mosquitoes from three independent cohorts. Vectorbase ID: Nasrat (AAEL008829), Closca (AAEL000961), Polehole (AAEL022628), and Nudel (AAEL016971). The mean ± SEM are shown as horizontal lines. Statistical significance is represented by stars above each column (unpaired Student’s t test; *** P < 0.001, NS not significant). A detailed phenotypic analysis can be found in Additional file 4: Table S3. Primers used are shown in Additional file 5: Table S4. RNAi-mediated knockdown efficiency was validated by quantitative real-time PCR (qPCR). Relative abundance of mRNA levels for Nasrat (E), Closca (F), Polehole (G), and Nudel (H) was analyzed in dissected mosquito ovaries at 36 h PBM. Mosquitoes were microinjected with each dsRNA at 4 days prior to blood feeding, as shown in Fig. 1. dsRNA-Fluc-injected mosquitoes were used as controls. A single mosquito analysis was performed to isolate total RNA, synthesize cDNA, and monitor silencing efficiency by qPCR. mRNA levels were normalized according to transcript levels of ribosomal S7 protein. Data are presented as MEAN ± SEM of 12 individual mosquitoes. *** P < 0.001 compared to RNAi-Fluc