The Interaction between a Sexually Transferred Steroid Hormone and a Female Protein Regulates Oogenesis in the Malaria Mosquito Anopheles gambiae
(A) Western blot under native nondenaturing gel condition using anti-20E and anti-MISO antibodies on atria of virgin or mated (8 hpm) females. Left and right arrowheads indicate 20E and MISO positive bands, respectively. (B) Co-immunoprecipitation of MISO and 20E in atria of virgin or mated (8 hpm) females. The anti-MISO immunoprecipitation (IP) was quantified with anti-20E ELISA. Mated atria showed a mean of 0.68 ng, while in virgin atria no 20E was detected. Three experiments were performed using a pool of 50 atria each, one-third used for the IP (upper panel) and two-thirds for the ELISA (lower panel). Data are represented as mean ± SEM. (C) MISO expression in atria dissected from females previously injected in the hemolymph with three 10-fold dilutions of 20E in ethanol (10% v/v) (starting from 2.5 µg of 20E per mosquito). Ethanol (10% v/v) and water-soluble cholesterol (0.25 µg/mosquito) were used as controls. Expression levels were measured 24 h after injection or mating, and were normalized to RpL19 and then to virgin levels in each replicate. A minimum of three replicates were performed for each condition. Data are represented as mean ± SEM. (D) qRT-PCR of MISO and Vg in females injected with dsEcR, analyzed at 6, 24, and 72 hpm. Expression levels (shown in logarithmic scale as fold changes relative to age-matched virgins) were normalized to the housekeeping gene RpL19. The analysis was performed in four replicates on pools of 5–10 female lower reproductive tracts. Data are represented as mean ± SEM. The asterisk indicates p<0.05.