1. High throughput testing for toxic and behavior modifying chemicals against Phlebotomine sand flies. Edgar D. Rowton, John P. Grieco*, Russell E. Coleman WRAIR, Silver Spring, MD; *USUHS, Bethesda, MD DoD Tri-Service Pest Mgmt Workshop, 13 Feb 2007

2. Background Leishmaniasis in Old World is vectored by sand flies (Diptera: Phlebotominae, Phlebotomus spp.) Iraq – PPMs, residual spraying not effective. Photo courtesy COL Peter Weina Photo courtesy LTC Russ Coleman Photo courtesy Ed Rowton

3. Objective Screen compounds for toxicity, contact irritancy (excito- repellency) and/or spatial repellency against sand flies. Existing compounds with known effect vs. mosquitoes - - DDT - - Permethrin - - α-cypermethrin Novel compounds - - SS220 - - DH220 - - DH5 - - Cmpd1 - - Cmpd2   Acetone (control) SS220 derivatives USDA proprietary plant derivatives

4. Methods Design assay chamber Prepare test compounds Conduct assays 1) 1) Toxicity assay 2) 2) Contact irritancy (exito-repellency) assay 3) 3) Spatial repellency assay

5. Modified assay chamber Grieco “High Throughput” modular assay chamber for mosquitoes. Grieco “High Throughput” modular assay chamber for mosquitoes. Grieco et al., J Am Mosq Control Assoc. 2005, Vol. 21. Test compound Control Spatial repellency assay Contact irritancy assay Chamber re-designed for sand flies to eliminate hiding places and secure test screen.

6. Prepare test screens Prepare test screens 25 nM/cm 2 test compound applied to nylon organdy material. Place insert into metal chamber for assay. Insert with treated screen

7. Assay #1 – Toxicity Sand flies exposed for 1 hour. Treated screen X ? Mortality determined at 1 & 24 hours.

8. Toxicity Assay

9. Assay #2 – Contact Irritancy Treated screen Sand flies exposed for 1 min. Butterfly door open for 10 min. X ?

10. Contact Irritancy Assay

11. Assay #3 – Spatial Repellency Treated screen Untreated screen ???? Choice assay. Sand flies released into middle unit, butterfly doors opened. After 10 min, count number and knockdown in each unit.

12. Spatial Repellency Assay (1) Sand fly movement (acetone only)

13. Spatial Repellency Assay (2) Sand fly knockdown

14. Summary   Re-designed assay chambers offer a useful tool for rapid screening of compounds for toxic, contact irritant and spatial repellent properties against sand flies.   Toxic / behavioral responses of sand flies were similar to mosquitoes.   Sand fly mortality was demonstrated for most of the compounds tested, e.g. permethrin, α-cypermethrin >> DDT.   All compounds demonstrated strong contact irritancy (excito- repellency).   Most compounds tested greatly deterred sand flies from entering the treated chamber. SS220 and USDA plant derivatives not only repellent, but also cause mortality within 24 hours. Ongoing research to develop repellents with this property that may also reduce population of biting insects. Future studies to determine repellency when attractant is present.

15. Acknowledgements Dr. Nicole Achée, USUHS Dr. Kamal Chauhan, USDA Elizabeth Broussard, WRAIR Mark Brown, USAMMDA Deployed War Fighter Protection (DWFP) Program

16. Questions? Disclaimer: Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense.