RNA interference-mediated knockdown of a GATA factor reveals a link to anautogeny in the mosquito Aedes aegypti

Abstract

Blood feeding tightly regulates the reproductive cycle in anautogenous mosquitoes. Vitellogenesis (the synthesis of yolk protein precursors) is a key event in the mosquito reproductive cycle and is activated in response to a blood meal. Before blood feeding, Aedes aegypti is in a state of reproductive arrest during which the yolk protein precursor genes (YPPs) are repressed. The regulatory region of the major YPP gene vitellogenin (Vg) has multiple GATA-binding sites required for the high expression level of this gene. However, a GATA factor (AaGATAr) likely acts as a repressor, preventing activation of this gene before a blood meal. Here we report in vivo data confirming the role of AaGATAr as a repressor of the Vg gene at the state of previtellogenic arrest. Using an RNA interference (RNAi)-mediated technique in conjunction with the Sindbis viral expression system, we show that knockdown of the AaGATAr gene results in an increased basal level of expression of the Vg gene and an elevated response to the steroid hormone 20-hydroxyecdysone in mosquitoes in a state of arrest. These experiments have revealed a component in the molecular mechanism by which anautogeny is maintained in A. aegypti.

Fig. 2.

(A) Schematic drawing of AaGATAr RNAi construct. (B) Schematic representation of the AaGATAr RNAi Sindbis virus and subgenomic RNAs expressed during infection. CAP, mRNA 5′ Cap Structure.

Fig. 1.

AaGATAr mRNA expression profile. Real-time PCR analysis was performed on cDNA samples obtained from mosquito fat bodies dissected at 12-h intervals during the previtellogenic state and either 2- or 4-h intervals during the vitellogenic state. cDNA was synthesized from total RNA from groups of five fat bodies per time point. Reactions were performed in triplicate. Data represent means ± SEM of triplicate samples.

Fig. 3.

Expression of the RNAi construct subgenomic RNA by Sindbis virus in infected mosquitoes. Five- to 7-day-old mosquitoes were infected with either control Sindbis virus (lacking insert) or Sindbis virus carrying the AaGATAr RNAi construct and allowed to incubate for 5 days at 28°C, 80% relative humidity. Wild-type mosquitoes are uninfected. Total RNA (7.5 μg) from each sample group was blotted and analyzed by Northern blotting by using a probe against the AaGATAr fragment used in the RNAi construct.

Fig. 4.

Vitellogenin expression is derepressed in AaGATAr RNAi-treated mosquitoes. Five- to 7-day-old mosquitoes were infected with either control Sindbis virus (lacking insert) or Sindbis virus carrying the AaGATAr RNAi construct and allowed to incubate for 5-6 days at 28°C, 80% relative humidity. Wild-type mosquitoes are uninfected. (A) Northern blot analysis of Vg expression in AaGATAr RNAi mosquitoes. Total RNA (7.5 μg) from each sample group was blotted and analyzed by Northern blotting by using a probe against vitellogenin. (B) Real-time PCR analysis of Vg expression in RNAi-treated mosquitoes. cDNA was synthesized from total RNA from groups of five fat bodies per time point. Reactions were performed in triplicate. Data were normalized by real-time PCR analysis of Actin levels in the cDNA samples. Data represent means ± SEM of triplicate samples. (C) Western blot analysis of Vg expression in AaGATAr RNAi mosquitoes. Total protein was extracted from groups of five mosquitoes. Twenty micrograms of total protein was blotted and probed with a monoclonal antibody against the 68-kDa small subunit of Vg.(D) Real-time PCR analysis of AaGATAr expression in RNAi-treated mosquitoes. Analysis was performed as in B. Data represent means ± SEM of triplicate samples.

Fig. 5.

The Vg gene is sensitized to the presence of 20E in AaGATAr knockdown mosquitoes. Five- to 7-day-old mosquitoes were infected with either control Sindbis virus (lacking insert) or Sindbis virus carrying the AaGATAr RNAi construct and allowed to incubate for 5 days at 28°C, 80% relative humidity. No virus treatments are uninfected wild-type mosquitoes. After 5 days, mosquitoes were injected with 0.5 μl of either ethanol (control) or 10^-6 M 20E. Vg expression was quantified by real-time PCR as described in Fig. 4B. Data represent means ± SEM of triplicate samples.