1. Use of the Fluorescent Tracer Technique (FTT) In the Laboratory and Field to Study Rodent-Sand Fly Interactions and Identify Potential Control Methods T.M. Mascari 1, E. Rowton 2, J. Clark 3, S. Gordon 3, L.D. Foil 1 1 Louisiana State University Agricultural Center, 404 Life Sciences Building, Baton Rouge, LA 2 Walter Reed Army Institute of Research, Silver Spring, MD 3 US Army Medical Research Unit – Kenya Unit 8900, Box 6801, DPO AE

2. Background - Phlebotomus papatasi and P. duboscqi are vectors of Leishmania major, agent of zoonotic cutaneous leishmaniasis (ZCL) - In arid foci, sand fly vectors are associated with rodent reservoirs of L. major - In Old World ZCL foci, rodent burrows are an immature habitat for P. papatasi - Sand fly larvae have been observed feeding on rodent feces -Adult female sand flies feed on blood -Adults of both sexes feed on sugars

3. There are many rodent/sand fly interactions Background

4. Sand fly marker system developed at LSU A) Answer important ecological questions - breeding sites for different sand fly populations - dispersal of adult sand flies B) Identify which insecticide applications would be most effective against a sand fly population - unlikely that all sand flies can be eliminated using a single control method C) Evaluate sand fly control measures - systemic insecticides - feed-through insecticides - insecticide-treated sugar baits Fluorescent Tracer Technique (FTT)

5. Fed rodents diet containing rhodamine B (RhB) Sand flies took bloodmeals from rodents Examined sand flies under fluorescence microscopy Sand flies that fed on RhB- treated rodents were marked FTT – Systemic insecticides T M Mascari 2008

6. Brightfield microscopy Fluorescence microscopy 5000 ppm0 ppm FTT – Systemic insecticides

7. What information could this give us? - identify extent to which sand flies in different rodent- sand fly interactions take bloodmeals from rodents targeted with baits - identify rodent-sand fly interactions that could be targeted with systemic insecticides - allow us to evaluate effects of rodent baits containing systemic insecticides on sand fly populations - sf infiltration from outside study area - natural pop. decline unrelated to control FTT – Systemic insecticides

8. Selection of systemic insecticides - palatable/non-toxic to target rodents - have persistent insecticidal effects Best candidates: Ivermectin Spinosad FTT – Systemic insecticides

9. Baringo District, Rift Valley Province, Kenya 2009 FTT – Systemic insecticides

11. Fed rodents RhB-treated diet Fed rodent feces to sand fly larvae Examined emerging sand flies under fluorescence microscopy Sand flies that fed on feces of RhB- treated rodents were marked FTT – Feed-through insecticides

12. Incandescent1 sec4 sec 5000 ppm 0 ppm 50 ppm 500 ppm FTT – Feed-through insecticides

13. ♂ ♀ 5000 ppm ♂ ♀ 0 ppm

14. What information could this give us? - identify whether sand flies in a certain rodent-sand fly interaction feed on the feces of rodents targeted with baits - identify what proportion of a sand fly population could be eliminated using feed-through insecticides - allow us to evaluate effects of rodent baits containing feed-through insecticides on sand fly populations. FTT – Feed-through insecticides

15. Feed-through insecticides that - are palatable/non-toxic to target rodents - are stable in the environment (persist in rodent feces once voided) Best candidates: Novaluron (persists up to 150 d in feces) Diflubenzuron Pyriproxyfen Ivermectin* FTT – Feed-through insecticides

16. A high proportion of sand flies feed on sugar and food dye sprayed on plants Food dyes don’t persist in sand flies (<20% were marked 3 dat) Rhodamine B and Uranine O are good alternatives to food dyes - don’t affect sf survival - persist for life of sand fly - can be used in combination (both dyes can be detected within a sand fly) FTT –Sugar baits

17. What information could this give us? - identify sand flies that feed on sugar baits sprayed on vegetation - information on ecology of sand flies (dispersal, etc.) - identify what proportion of a sand fly population could be targeted with insecticide-treated sugar baits - our lab currently is evaluating insecticide sugar baits Use an insecticide with low toxicity against non-target animals – boric acid has been used against mosquitoes FTT –Sugar baits

18. Summary FTT allows us to determine which sand fly populations could be susceptible to feed- through insecticides, systemic insecticides, or insecticide-treated sugar baits In the event that a treatment doesn’t work, FTT allows us to establish reasons why and to plan future research accordingly

19. Acknowledgements This work was supported by a grant from the Deployed War-Fighter Protection (DWFP) Research Program, funded by the U.S. Department of Defense through the Armed Forces Pest Management Board (AFPMB).