Fig. 6

Nasrat, Closca, Polehole, and Nudel are essential for eggshell melanization and oocyte membrane permeability. A An in vitro follicle melanization assay was conducted using follicles isolated from gravid RNAi mosquitoes at 96 h PBM. Timing of dsRNA microinjection and blood feeding schedule was identical to those shown in Fig. 1. Follicles were photographed 5, 90 and 150 min after dissection. B Each bar corresponds to mean egg melanization (%) from five individual mosquitoes. 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, ** P < 0.01). C An in vitro follicle melanization assay was performed using a protease inhibitor cocktail (PI) to determine the role of proteases on eggshell melanization. The follicles were photographed 5, 90, and 150 min after follicle dissection. Follicles were incubated with PI (1X) at 0, 10, or 20 min after follicle dissection. Follicles were also incubated with PMSF (10 mM) immediately after dissection. D Each bar corresponds to mean egg melanization (%) from five individual mosquitoes. 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). E Follicle permeability assays were conducted using a Rhodamine B marker, with cellular uptake of Rhodamine B observed in primary follicles possibly due to a defective eggshell and oocyte plasma membrane. A detailed phenotypic analyses are shown in Additional file 8: Table S7 and Additional file 9: Table S8