Posttranscriptional control of the competence factor betaFTZ-F1 by juvenile hormone in the mosquito Aedes aegypti

Abstract

In anautogenous mosquitoes, vitellogenesis, which includes production of yolk protein precursors, requires blood feeding. Consequently, mosquitoes transmit many diseases. Understanding the molecular mechanisms of vitellogenesis regulation will contribute significantly to vector control strategies. Newly emerged Aedes aegypti females require 3 days before becoming competent to activate vitellogenesis in response to a blood-meal-initiated, elevated titer of 20-hydroxyecdysone (20E). An orphan nuclear receptor gene betaFTZ-F1 is transcribed in the fat body of newly emerged mosquito females; however, the betaFTZ-F1 protein is only found 3 days later. Dramatically increased titer of the juvenile hormone III (JH III) is essential for the acquisition of 20E competence. In vitro fat body culture experiments have shown that betaFTZ-F1 protein appears after exposure to JH III. Injection of double-stranded RNA complementary to betaFTZ-F1 into newly emerged females attenuated expression of the early genes EcR-B, E74B, and E75A and the target YPP gene Vg, in response to a blood meal. Thus, betaFTZ-F1 is indeed the factor defining the acquisition of competence to 20E in the mosquito fat body. Moreover, this is achieved through JH III-mediated posttranscriptional control of betaFTZ-F1.

Fig. 1.

20E responsiveness in the fat body of previtellogenic female mosquitoes. (A) Hormonal titers during the first vitellogenic cycle of the anautogenous mosquito, A. aegypti. BM, blood meal; E, eclosion; JH III titers [modified from Shapiro et al. (18)]; 20E titers [modified from Hagedorn et al. (6)]. (B) Fat bodies from mosquitoes were isolated at 12-h intervals after eclosion and cultured in vitro for 6 h in medium with or without the presence of 1 × 10^-6 M 20E. The expression of the genes involved in the 20E signal pathway was determined by real-time PCR and was normalized to β-actin expression. Representative data (mean ± SEM) from at least three independent experiments are shown. CM, culture medium.

Fig. 2.

JH III augments activation of the Vg gene by 20E. Fat bodies from newly emerged female mosquitoes were exposed to 1 × 10^-5 M JH III (♦) or acetone (□) in vitro for 12 h, followed by culture in the presence of 1 × 10^-6 M 20E. Fat bodies were also incubated directly with 20E as a control (▴). The expression of the indicated genes was determined by real-time PCR and was normalized to β-actin expression. Arbitrary units are plotted against time of incubation in medium with 20E. Representative data (mean ± SEM) from at least three independent experiments are shown.

Fig. 3.

Mosquito βFTZ-F1 protein in the fat body during vitellogenesis. (A) Fat-body nuclear extracts were separated on SDS/10% polyacrylamide gel, transferred to polyvinylidene fluoride membrane, and analyzed with rabbit polyclonal anti-AaβFTZ-F1 antiserum. The antibodies bound were then stripped, and the membrane was reprobed with monoclonal antibody against β-actin. TNT, in vitro synthesized AaβFTZ-F1. (B) βFTZ-F1 mRNA in the fat body of adult females was measured by real-time PCR. Representative data (mean ± SEM) from at least three independent experiments are shown. (C) βFTZ-F1 in the fat-body nuclear extracts was examined with electrophoretic gel mobility shift assay as described by Li et al. (22). The arrowhead indicates the supershifted band. F1RE, consensus binding site of βFTZ-F1; FBNE, fat-body nuclear extracts.

Fig. 4.

JH III stimulates synthesis of βFTZ-F1 in fat bodies cultured in vitro. Fat bodies of newly emerged female adults (6 h PE) were cultured in medium for 18 h in the presence of JH III or acetone. Western blot analyses were performed on fat-body nuclear extracts by using antibodies against AaβFTZ-F1 and β-actin. Fat bodies taken directly from female mosquitoes at 6 h PE and 72 h PE were used as controls. Representative data from three independent experiments are shown. CM, culture medium.

Fig. 5.

The effect of RNAi on βFTZ-F1 expression in A. aegypti. (A) Schematic diagram of nuclear receptor βFTZ-F1 showing the A/B domain that was used to generate complementary dsRNA. (B) dsRNAs were injected into thoraces of female mosquitoes as described in Materials and Methods. βFTZ-F1 mRNA was measured at the indicated time after blood feeding by real-time PCR and was normalized to β-actin expression. Representative data (mean ± SEM) from at least three independent experiments are shown. ▴, uninjected A. aegypti Rockefeller/UGAL strain; □, injected with dsRNA complementary to malE; ♦, injected with βFTZ-F1 dsRNA. (C) βFTZ-F1 protein in unfed mosquitoes at 84 h PE detected by Western blot analyses. The signals corresponding to βFTZ-F1 were measured with VersaDoc (Bio-Rad) and normalized to the actin-loading control. βFTZ-F1 proteins in mosquitoes injected with control dsRNA and βFTZ-F1 dsRNA were 95% and 9%, respectively, of those in untreated female mosquitoes. TNT, in vitro synthesized βFTZ-F1 protein.

Fig. 6.

Expression of 20E-induced genes in mosquitoes subjected to βFTZ-F1 RNAi. (A) The transcripts of indicated genes in female mosquitoes were measured after blood feeding by real-time PCR and were normalized to β-actin expression. Arbitrary units are plotted against developmental time in hours PBM. Representative data (mean ± SEM) from three independent experiments are shown. Open bars, uninjected A. aegypti Rockefeller/UGAL strain; shaded bars, injected with dsRNA complementary to malE; solid bars, injected with βFTZ-F1 dsRNA. (B) Appearance of Vg protein in fat body was significantly inhibited by βFTZ-F1 RNAi. Proteins were extracted from mosquitoes at 84 h PE and 24 h PBM and were analyzed with monoclonal antibody against the Vg small subunit. The two bands represent different posttranslational modifications of the small subunit.