Update on the search for new toxicants and methods of control James J. Becnel

1. Update on the search for new toxicants and methods of control James J. Becnel 1. Novel insecticide chemistries or formulations 2. Personal protection • Application technology

2. Toxicological evaluation of compounds • Cooperative Project with Chemical Companies • Cooperative Projects with ARS Natural Products Utilization Research Unit (NPURU)

3. Screening Protocols • Phase 1 –chemical screening using high throughput Ae. aegypti assay with 1st instar larvae and categorize activity based on 24 hour mortality • Phase 2 -Compounds >80% mortality evaluated with topical assays (hit rate of ~ 10%) • Phase 3 -Determine LD50 values for the most active adulticides and LC50 values for the most active larvicides

4. High throughput screen • Day 1: Hatch Aedes aegypti eggs • Day 2: Sort 1st instar larvae to 24-well plates, 5/well Add 1ml of water and food Add 10 ul of insecticide solution • Day 3: Score bioassay data (dead/total)

5. Phase 2 - Topical application Maximum capacity: < 200 chemicals per week Small Amount Fast Accurate Easy

6. Diversity Samples from Chemical Library Chemical

7. Phase 1 Results for 9344 compounds 8.8% hit rate

8. Chemtura Summary Four samples advanced to test against permethrin-resistant strain Puerto Rico:C2395,B6572, C3506, and F9842 C2395 active at 100 ppm, B6572 active at 25 ppm C3506 and F9842active at 12.5 ppm against adults

9. ARS Natural Products Utilization Research Unit, Oxford, MS Charles Cantrell David Wedge Kumudini Meepagala

10. Results: Chemicals from plants NPURU

11. Chemical approach: Summary • Employed high throughput screening larvae assay and screened ~10,000 compounds; additional ~2,000 by end of year. • Two compounds with good adult activity, follow-up in progress • Screened hundred of natural plant chemicals; several showed high adulticidalactivity

12. Molecular Pesticide Development Through RNAi Technology Molecular pesticide Pesticide whose active ingredient is RNA.

13. Approach for the Development of Molecular Pesticides Target Essential Pathways Molecular Cloning (DNA, RNA, PCR) Programmed Cell Death, Mitochondria Pathway, etc (Aedes aegypti model) Vector, Carrier, Formulation Molecular Pesticide Construction (Aedes aegypti model) Bioassays

14. “Pesticidal Double Stranded RNA Composition and Method of Use Thereof,” Patent Application Pridgeon, Becnel and Strickman Current Status of Application as of Nov. 9, 2009 Received a restriction requirement from the Patent Office Estimate that within 6 months we'll receive a First Office Action on the merits

15. Status of IAP RNAi constructs as pesticides • In selection process of a single IAP construct for mass-production. Contract in place (1-25-2010) to make 5 grams of IAP RNAi constructs. • Investigating additional IAP targets (IAP2, IAP3, IAP4) • Probed 44 regions of four IAP genes for activity • 2640 of 7986 bp total (~32% of sequence info) • Activity of these constructs being evaluated IAP 1 IAP 2 IAP 3 IAP 4

16. Control Pesticide-treated mRNA cDNA synthesis Rsa I digestion & adaptor ligation mRNA cDNA synthesis Rsa I digestion & adaptor ligation cDNA hybridization/subtraction TA cloning Sequencing Annotation & QPCR PCR-select subtractive cDNA library A revolutionary method for finding differentially expressed genes Very well-suited for the identification of rare critical transcripts, which are typically the most difficult to obtain

17. Subtractive library clone designations Orlando permethrin Puerto Rico permethrin Puerto Rico minus induced induced Orlando P450 Hypothetical/Unknown Energy Housekeeping Other Insecticide resistance Three P450 pulled out of Puerto Rico minus Orlando subtractive library. Greater number of metabolism (energy) genes in Puerto Rico than Orlando.

18. P450 screening- ongoing and future goals • Designed and generated dsRNA constructs to knock-down P450 genes. • Currently determining ability to knock-down P450 gene expression (time to knock-down and duration). • Functional studies with permethrin treatments to determine if specific knock-down of P450s increases susceptibility in Puerto Rico. • Assess specificity using other mosquito spp.

19. Molecular approach: Summary • Refining IAP constructs to select those with high efficacy for large scale production. • Molecular targets were identified by subtractive cDNA libraryafter challenging the mosquitoes with pesticides. • Functional role of several P450s implicated in permethrin resistance to be evaluated.

20. DWFP Toxicology Team Members • Dr. Julia Pridgeon (Toxicologist & Molecular Biologist) • Dr. Liming Zhao (Molecular Biologist) • Dr. Monique Coy (Toxicologist & Molecular Biologist) • Bill Reid (Technician, Toxicology & Molecular Biology) • Neil Sanscrainte (Technician, Toxicology & Molecular Biology) • LT Anne Thornton (visiting scientist, Toxicology & Molecular Biology) All members in the Mosquito and Fly Research Unit Center for Medical, Agricultural, and Veterinary Entomology ARS, USDA and the Deployed War Fighter Protection Research Program

21. Monique Bill Kelly Neil