1. Project objectives: 1) Screen Bt toxins for activity against Diaprepes root weevil (DRW); 2) Identify the most effective dsRNA constructs against DRW; 3) Assess the combined action of dsRNA and Bt toxins; 4) Assess four Bt transgenic citrus lines for DRW resistance. Objective 1. Bacterial Pesticidal Proteins: During this first reporting period, 11 of some 15 bacteria-derived pesticidal proteins drawn from four different structural groups were expressed in Bonning’s lab by use of either E. coli- or Bacillus thuringiensis-based expression systems. Pesticidal proteins were harvested from E. coli or following Bt sporulation, purified and solubilized as required using standard procedures. Cry proteins were trypsin activated. These proteins are now ready for testing in initial bioassays against DRW. DRW Colony: Meanwhile, in Stelinski’s lab, a new colony of DRW has been established to generate insects for this project. The culture was initiated from adult weevils collected from citrus groves in central Florida in 2023. Larvae are reared on an artificial diet developed by Beavers (1982) using procedures described by Lapointe and Shapiro (1999).DRW Bioassay: To establish a reliable bioassay protocol for testing of bacterial pesticidal proteins against DRW larvae, soil-column, seedling, and meridic diet bioassay methods were compared using a proxy formulation of B. thuringiensis subsp. tenebrionis (Btt; CX-2330 85% [AI]). Bioassays were conducted to evaluate survival of DRW neonates and 5-week-old larvae after exposure to bacterial suspensions of Btt. While all bioassays indicated activity of the Btt treatment as compared with the control (particularly against neonate larvae), the meridic diet method produced the most consistent results with the least mortality observed in untreated control treatments. Moreover, this method was the easiest to establish, and the least expensive in material costs and time investment. While certain experiments may require use of other bioassay methods or variations thereof, these initial results indicate that the meridic diet bioassay will serve our needs for testing of bacterial pesticidal proteins. Objective 2. Gene Silencing RNAs: the gene silencing RNAs (dsRNAs) and primers have been designed for all DRW target genes by Killiny, and reagents necessary for dsRNA synthesis are on order. Citations: Beavers, J. B. 1982. Biology of Diaprepes abbreviatus (Coleoptera: Curculionidae) reared on an artificial diet. Florida Entomologist. 65: 263-269.Lapointe, S. L., & Shapiro, J. P. 1999. Effect of soil moisture on development of Diaprepes abbreviatus (Coleoptera: Curculionidae). Florida Entomologist, 82: 291-299. 2. Plans for the next quarter:Objective 1: Initiate screening of bacterial pesticidal proteins for toxicity against DRWObjective 4: Conduct bioassays to assess the survival of DRW on transgenic plants that express bacterial pesticidal proteins. 3. Budget status: Hiring of the postdoctoral researcher for work on objective 2 in Killiny’s lab has been delayed. Otherwise the project is on track.