1. Techniques for Evaluating Insecticide Resistance in Filth Flies R esistance Profiles: Which Test To Use? Phil Kaufman Entomology and Nematology Dept. University of Florida

2. Background  Resistance is genetic, therefore inherited –Return to susceptibility not guaranteed  Speed of return is quite variable  Resistance is a population phenomenon –All sampling methods are biased –All testing methods are biased –We must identify our needs…. and choose according to our abilities

3. Susceptible Individuals Predominate Insecticide Application

4. Resistant Individuals Predominate Susceptibles still present, few in number

5. How to evaluate?  Biochemical/Immunological (Genotypic) methods –Molecular tools targeting physiological processes –Genetic mapping - Sequencing/gene expression  Phenotypic methods –Exposure followed by death counts

6. Treat and Count  Technical or Formulated? –Technical much more precise  Very precise results  Eliminate effect of formulation shifts  Eliminate concentration problems of formulations –Formulated often much easier  Loss of precision - more variables  Can compare different formulations  Provides a “real world” result

7. Insect issues…..  Lab reared or Field collected? –Lab provides a more even comparison  No parasites, healthy  Similar sized/aged organisms  Genetic bottleneck? –Field  No rearing issues  Faster turnaround  Variable size/age/health

8. Insect issues….cont.  What stage to use? –Often the stage that is pestiferous - Adult  Insect sex –Important differences between sexes  Size, metabolism, longevity  Insect age –Can greatly influence survival

9. Insect issues….cont.  Insecticide susceptible strain? –Where to get one? –What is your testing interest?  LD-50 or Resistance Ratio (RR)  Colony? –Who keeps one for your insect? –Use WHO standard?  No Susceptible comparison…. –Reduces comparative values –Alright if using a diagnostic dose?

10. Testing methods - Direct vs. Indirect  Direct –Microapplicator  Tiny drop applied to individual insects  Cumbersome - Most accurate –Dip method  Insect submerged into a solution of known strength

11. Testing methods - Direct vs. Indirect  Indirect –Treated glass  Most common and often best method –Treated organics  Treating filter papers, cloth, wood  May have adsorption issues, especially formulated

12. Treated Board Studies

13. Holding patterns….  How long to expose insects to insecticide? –1 hr, 4 hr, 24 hr? –Irrelevant - pick one, dose will be adjusted  When to count the dead? –Post-treatment count? OR –24 or 48 hr after exposure?  Allows for kdr-resistant resistance detection.

14. Resistance Profile vs. Diagnostic Dose  Resistance Profile –Profile requires increasing doses  Usually predates use of Diagnostic Dose  Single dose or Diagnostic dose –Often used with formulated materials  Label Rate or a Given level exposure  Replication vs. Psuedoreplication –All steps must be repeated  Includes weighing and mixing all solutions

15. Analysis  Control mortality –Critical  Many blood-feeding Diptera survive poorly off host –Often use “Abbott’s Formula”  Probit (logistic regression) –Generates mortality line –Used for Lethal Dose (LD) calculation  Often used to identify 50% or 99% mortality value (% Mortality - % Control Mortality) (1 - % Control Mortality) = * 100

16. Real-world Data  House flies from 8 NY poultry and 4 dairy farms surveyed  Many formulations tested –Multiple active ingredients  Residual contact, feeding and larvicide assays  Take home: Variations between sites, chemicals, methods, insecticide source….

17. Experimental Compound

18. Methomyl (Feeding)

19. Dimethoate

20. Tetrachlorvinphos

21. Cyfluthrin

22. Permethrin

23. Permethrin

24. Tetrachlorvinphos

25. Dimethoate

26. Ectiban EC

27. Ectiban WP

28. Which one to use? Needs based.  To rapidly identify resistance –Field collected**** –Lab reared  Precision –Technical**** –Formulated  Deployed situations –Technical –Formulated****  Control failures –Technical –Formulated****

29. www.who.int/whopes/resistance/en/ WHO - Test Kits

30. Purchasing Kits

31. Filth Flies