1. Progress in DWFP and Related Research Ulrich R. Bernier USDA-ARS-CMAVE Mosquito & Fly Research Unit 352-374-5917 ubernier@gainesville.usda.ufl.edu 2007 DoD Pest Management Workshop 12-16 February 2007

2. DWFP Projects to be Discussed  Chemical structure activity studies  Development of portable devices  Biting protection assessment of uniforms  Development of inhibitor sustained release delivery systems  Laboratory and field studies with sand flies in Egypt

3. Structure-Activity Studies of Known Arthropod Repellents and Toxicants Using CODESSA and QSAR Collaboration: Agreement with University of Florida Department of Chemistry Research problem: Characterize the molecular structural and electronic properties that: –correlate with level of repellent activity against multiple insect species –lead to insecticidal activity Key point: Since 1942, USDA has compiled records for thousands of compounds tested for repellent and insecticidal properties. Military relevance: Development of novel repellents and insecticides will enhance the arsenal of products to control insects that transmit disease to personnel.

4. Candidate repellent in vivoin vitro Topical cage test Cloth patch cage test GC/MS Purity Spatial inhibition HITSS Olfactometer MFRU Repellent Testing Paradigm Toxicology tests Product Commercial partner Registration Candidate toxicant Toxicant track Field evaluation Also tested with infected mosquitoes

5. Experimental Classes of Repellents Used in Models Duration of protection On cloth patchOn skin Class 1ineffective0-60 min Class 21-5 days61-120 min Class 36-10 days121-180 min Class 411-20 days181-299 min Class 5=> 21 days=> 300 min

6. Class 1 11 (50%) 4 Class 24 2 (14%) 1 Class 377 3 (50%) 12 Class 412 1 (50%) Class 5 1 (33%) Class 1Class 2Class 3Class 4Class 5 Experimental Classes Predicted Classes Model-Predicted Classification vs. Experimental Classification of Repellents

7. Carboxamides and Piperidines DEET Piperidine 1-(3-methylbenzoyl)piperidine

8. Bioassay of Synthetic Piperidines as Candidate Attraction-Inhibitors (Candidates are coded P1-33) USDA InhibitorDeetAttractant Blend P13 AI3-38739 P6 3-Methyl AI3-33510

9. Current database contains over 30,000 compounds tested by USDA Spatial repellency has been assayed with the olfactometer for the 33 synthetic piperidines. Analysis of the carboxamides will take place in 2007. Work is in progress on 5 database sets: –House fly residual insecticides –Anopheles spp. larvicides –Ae. aegypti repellents –Anopheles spp. residual insecticides –Oc. taeniorhyncus insecticides Repellents assays (cloth patch test) completed for 6 piperidines- both CPT and MED have been determined—the remaining 27 piperidines and carboxamides should be completed by 30 Jun 2007. Structure-Activity Studies (cont.)

10. Collaboration: Agreement with University of Florida Dept. of Chemistry Research problem : An economical field device for airborne detection and quantification ULV spray fate and dispersal, repellents, and inhibitors is not currently available. Key point: FAIMS devices: –are small, low-cost units –exhibit high chemical sensitivity and selectivity with low noise and can separate isomers –can be used for continuous monitoring and work at atmospheric pressure Military relevance: Addition of a low-cost, field deployable device to mask attraction of personnel from biting insects can result in reduced disease risk to deployed personnel. Development of Portable Devices for Detection and Quantification of Insecticides, Repellents, and Inhibitors

11. Field Asymmetric Ion Mobility Spectrometry (FAIMS)

12. Field-Deployable Devices

13. Preliminary results with naled and malathion have demonstrated that this technique can separate isomers and be used in the field. Currently exploring FAIMS optimization of naled, malathion, permethrin, d-phenothrin, resmethrin, bifenthrin, cyfluthrin, and deltamethrin Next step is the evaluation of FAIMS as a stand-alone detector and determination of detection limits for target compounds Development of Portable Devices (cont.)

14. Assessment of Biting Protection of U.S. Military Uniforms and Development of Criteria to Protect Deployed Troops in the Future Collaboration: Agreement with Natick Soldier Center—MIPR initiated by USMC Research problems: –Determine if application of permanent press finish affected performance of permethrin-treated Marine Corps Combat Utility Uniforms (MCCUUs) –Develop a protocol to determine biological efficacy of factory- treated MCCUUs against mosquitoes Key point: DWFP component of this work is to establish baseline efficacy data of sand fly bite protection from factory-treated MCCUUs. Military relevance: Factory-treated MCCUUs that retain biologically efficacious levels of permethrin over their lifetime preclude the need for field-treatment of uniforms and may ultimately reduce disease risk.

15. Untreated sleeve - Female mosquitoes land and probe in search of a blood meal

16. Treated sleeve after 2 min - Many females are still on the sleeve but few obtain blood

17. Treated sleeve after 8 min - Few mosquitoes remain on the sleeve and many have left the garment

18. American Apparel First Article Quantitation of Permethrin in Fabric by Gas Chromatography (Natick Soldier Center) Treated MCCUUs in 2004 (phase 2) exhibited rapid initial losses of permethrin when laundered. 0 washes20 washes 50 washes

19. Assessing Biting Protection in Uniforms (cont.) Summary –Initial assessment of fabrics started on March 17, 2006 –Three companies have qualified, one is undergoing first article validation –There have been three failures, one company has failed twice Future –Bite protection for first article validation will be completed by 10 Mar 2007 –Continue MCCUU evaluation as needed –ACU study involving field treatment –Evaluation of factory-treated MCCUU field performance (with JAX NECE, CDR Rankin and LT Doud at Lejeune) –Development of new treatments – spatial effect

20. Development and Testing of Sustained Release Delivery Mechanisms for Repelling Mosquitoes and Biting Insects Collaboration: Agreement with Bioguard R&D to develop delivery devices for field release of attraction-inhibitors Research problem: USDA-developed attraction inhibitors require encapsulation or binding of active ingredients in suitable non-reactive media to: –produce a sustained release at efficacious delivery rates –to facilitate transport to the field where they will produce repellency in the field for 30 days. Key point: Inhibitors that prevent host detection by biting insects are a novel method of personal protection technology. Military relevance: Improvement of insecticide application technology, personal protection and vector inhibitors used in the field can yield more effective protection of deployed personnel and result in reduced disease risk.

21. Volatile samples are contained in pressurized canisters and delivered into an olfactometer port to test mosquito attraction or inhibition

22. Kairomones alert mosquitoes that a host is near. Mosquitoes follow the plumes upwind (anemotaxis) toward the source of the odor. Adapted from: Bernier, U.R., Laboratory Research and Development of Attractants, Inhibitors and Repellents, Tech. Bull. Florida Mosq. Control Assoc., 2006, 9:9-16.

23. Many of the less volatile repellents, like DEET, are a feeding deterrent or contact irritant that prevent biting. The chemical on the skin surface is unappealing for the mosquito to land, remain on, or feed through. Adapted from: Bernier, U.R., Laboratory Research and Development of Attractants, Inhibitors and Repellents, Tech. Bull. Florida Mosq. Control Assoc., 2006, 9:9-16.

24. An attraction-inhibitor decreases host-finding ability by masking or cloaking the kairomones, producing anosmia or hyposmia in the mosquitoes. ? Adapted from: Bernier, U.R., Laboratory Research and Development of Attractants, Inhibitors and Repellents, Tech. Bull. Florida Mosq. Control Assoc., 2006, 9:9-16.

25. Addition of a small amount of inhibitor to an attractant blend or to human emanations decreased attraction of mosquitoes*

26. Bioassay of Candidate Spatial Repellents for Use in Field-Deployed Devices to Cloak Humans from Mosquitoes Attractant Blend Efficient Inhibition

27. Examples of carriers

28. Nanoclay containing attraction- inhibitors Air-activated warmer to elevate and maintain a constant temperature Outer packaging of durable material to contain the system Development of Field Kits to Deliver Sustained Release of Attraction-Inhibitors

29. Second version of prototype release devices Package size approximately 3” x 3”

30. Current device to be tested in Egypt. It delivers 40- 80 mg/day for 3-5 days at wind speed of 3-5 mph and provides coverage up to 10-15 m from the device. Capsule is 1.5” diameter, 0.5” height.

31. Advantages of prototype devices: –Predicted release of active ingredients at efficacious levels for 30+ days at 1 m from source and 3-5 days at 10- 15 m from source –Next generation device is easily transportable to field in glass ampule –Packaging meets DOT and IATA requirements for unregulated transport Final designs for laboratory and field testing are related to individual personnel protection (IPP) requirements. Future: –Preliminary design and evaluation of the broad area pack (BAP) –Design and evaluation of nano-carrier for permethrin-based fiber systems for extended release-based longevity Development of Sustained Release Mechanisms (cont.)

32. Laboratory and Field Experiments with Attractants, Inhibitors and Repellents against Sand Flies in Egypt Collaboration: LCDR David Hoel, NAMRU-3, Cairo, Egypt Research problems: –Explore chemical and physical means to enhance collections of sand flies in commercial field traps –Explore inhibition of sand fly host-seeking in the laboratory and field. –Develop baseline efficacy of MCCUUs against sand flies. Key point: NAMRU-3 conducts field experiments in Aswan—>90% Phlebotomus papatasi Military relevance: Permethrin-treated uniforms are less protective against sand fly bites than against mosquitoes. Development of novel personal protectants for use against sand flies will improve protection of troops.

33. Spacious laboratories for visiting scientists

34. Office space for the taking

35. Field Site in Bahrif, Aswan

36. LCDR Hoel working on a different trap study

37. Jerry Hogsette conducting research with fly traps

38. Trap site 1 - Typically the most productive site

39. Sample acquisition Human hair as one of the treatments

40. 1-Octen-3-ol lure and “human” lure

41. Baited trap- Traps are operated overnight (~1700 to 0700 hrs)

42. Trap site 4 Measuring overnight loss of lure

43. Insects/Night 38 32

44. Laboratory and Field Experiments in Egypt (cont.) Three sets of field data have been collected August- September, 2006). No significant difference observed between trap lures. In future laboratory studies inhibitors will be tested against sand fly species in colonies at NAMRU-3 Future field studies will involve: –exploration of trap location and physical arrangement –examination of best LED-lure-placement combination –incorporation of inhibitors in experiments –determination of daytime resting sites of sand flies