Optimize Effector Genes For Population Replacement And Reduction.

Specific Aim 1: Characterization of Aedes aegypti promoters.  We will adapt the GAL4/UAS system to screen efficiently the activity of a number of gene promoters. We expect to have at the end of this work multiple optimized promoters for expressing effector molecules. The GAL4/UAS system was developed such that the Gal4 protein, expressed from a specific promoter, binds to UAS, stimulating expression of a second (reporter or effector) gene only in those cells in which Gal4 is expressed.  Transgenic lines are being made with reporter (UAS-luciferase or UAS-EGFP) or effector genes, and a second set with promoter/Gal4 constructs.  Mating these lines completes the GAL4/UAS system and allows analysis of promoter activity and effector expression. The promoter of the fat body-expressed gene, vitellogenin, has been well characterized already, and we will research genes expressed in other tissues infected by DVs. Midgut-specific promoters will be analyzed from genes expressing a carboxypeptidase, glutamine synthetase, ferritin, and peritrophic matrix protein.  The D7 and Apyrase gene promoters will be adapted for salivary gland-specific expression. The corresponding 3'-end untranslated regions (UTR) as well as the promoter and 5’UTR will be included as they may contain regulatory elements. In addition, a female-specific promoter from the Aedes Actin-4 gene (AeAct-4), and a sex-specific splicing system from transformer or doublesexgenes will be identified to allow sex-specific production of a protein from a non-sex-specific promoter. 

Specific Aim 2: Development of smart effector genes blocking DV transmission.  Multiple effector gene strategies will be researched to ensure that transgenic mosquitoes expressing optimized effector genes are available.  RNAi-based effectors- RNA interference (RNAi) is an ancient, homology-dependent, gene-silencing system, triggered by double-stranded RNA (dsRNA). Functional homologues of RNAi components have been characterized in mosquitoes.  RNAi-based effectors must be transcribed in the same cells as the target sequence.  The use of a 500bp dsRNA trigger should preclude RNA virus escape by generating an array of 21-23 nucleotide small interfering RNAs that effectively target fast evolving RNA virus genomes.  Many RNA viruses express suppressors of RNAi; however, no RNAi suppressors have been identified yet in flaviviruses. Inherent challenges with this strategy can be overcome by early, robust expression of the effector gene as, or before, the virus enters the mosquito (Specific Aim 1). Peptide–based effectors showed that peptides recognizing mosquito tissue surface proteins blocked entry of a malaria sporozoite into the salivary glands of a transgenic mosquito. The challenge here is to identify effector proteins that block DV transmission yet can be effective against a rapidly evolving RNA virus. An alternate approach is express at the wrong stage of development a peptide that affects mosquito behavior. Completion of this Specific Aim will provide RNA-based effector genes that block DV transmission when expressed in transgenic mosquito midguts and salivary glands, and peptide-based effector genes that block DV transmission or prevent multiple rounds of mosquito feeding.

Specific Aim 3: Development of RIDL effector genes. Effector genes will be developed for Ae. aegypti that optimize population suppression using RIDL.  RIDL effector genes combines with the appropriate promoters (Specific aim1) will allow tissue-, sex- and life stage-specific lethality. Completion of this Specific Aim will yield RIDL strains that can be used to control dengue transmission.

Objective 1                          Objective 2                         Objective 3