techniques for evaluating insecticide resistance in filth flies r esistance profiles: which test to...
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Techniques for Evaluating Techniques for Evaluating Insecticide Resistance in Insecticide Resistance in
Filth FliesFilth Flies
RResistance Profiles: Which Test To esistance Profiles: Which Test To Use?Use?
Phil KaufmanPhil Kaufman
Entomology and Nematology Entomology and Nematology Dept.Dept.
University of FloridaUniversity of Florida
BackgroundBackground
Resistance is genetic, therefore Resistance is genetic, therefore inheritedinherited– Return to susceptibility not guaranteedReturn to susceptibility not guaranteed
Speed of return is quite variableSpeed of return is quite variable
Resistance is a population Resistance is a population phenomenonphenomenon– All sampling methods are biasedAll sampling methods are biased– All testing methods are biasedAll testing methods are biased– We must identify our needs…. and choose We must identify our needs…. and choose according to our abilitiesaccording to our abilities
Susceptible Individuals Susceptible Individuals PredominatePredominate
InsecticideApplication
InsecticideApplication
Resistant Individuals Resistant Individuals PredominatePredominate
Susceptibles still present, Susceptibles still present, few in numberfew in number
How to evaluate?How to evaluate?
Biochemical/Immunological Biochemical/Immunological (Genotypic) methods(Genotypic) methods– Molecular tools targeting Molecular tools targeting physiological processesphysiological processes
– Genetic mapping - Sequencing/gene Genetic mapping - Sequencing/gene expressionexpression
Phenotypic methodsPhenotypic methods– Exposure followed by death countsExposure followed by death counts
Treat and CountTreat and Count
Technical or Formulated?Technical or Formulated?– Technical much more preciseTechnical much more precise
Very precise resultsVery precise results Eliminate effect of formulation shiftsEliminate effect of formulation shifts Eliminate concentration problems of Eliminate concentration problems of formulationsformulations
– Formulated often much easierFormulated often much easier Loss of precision - more variablesLoss of precision - more variables Can compare different formulationsCan compare different formulations Provides a “real world” resultProvides a “real world” result
Insect issues…..Insect issues…..
Lab reared or Field collected?Lab reared or Field collected?– Lab provides a more even comparisonLab provides a more even comparison
No parasites, healthyNo parasites, healthy Similar sized/aged organismsSimilar sized/aged organisms Genetic bottleneck?Genetic bottleneck?
– FieldField No rearing issuesNo rearing issues Faster turnaroundFaster turnaround Variable size/age/healthVariable size/age/health
Insect issues….cont.Insect issues….cont.
What stage to use?What stage to use?– Often the stage that is pestiferous - Often the stage that is pestiferous - AdultAdult
Insect sexInsect sex– Important differences between sexesImportant differences between sexes
Size, metabolism, longevitySize, metabolism, longevity
Insect ageInsect age– Can greatly influence survivalCan greatly influence survival
Insect issues….cont.Insect issues….cont. Insecticide susceptible strain?Insecticide susceptible strain?
– Where to get one?Where to get one?– What is your testing interest?What is your testing interest?
LD-50 or Resistance Ratio (RR)LD-50 or Resistance Ratio (RR)
Colony?Colony?– Who keeps one for your insect?Who keeps one for your insect?– Use WHO standard?Use WHO standard?
No Susceptible comparison….No Susceptible comparison….– Reduces comparative valuesReduces comparative values– Alright if using a diagnostic dose?Alright if using a diagnostic dose?
Testing methods - Direct Testing methods - Direct vs. Indirectvs. Indirect
Direct Direct – MicroapplicatorMicroapplicator
Tiny drop applied to Tiny drop applied to individual insectsindividual insects
Cumbersome - Cumbersome - Most accurateMost accurate
– Dip methodDip method Insect submerged into a Insect submerged into a solution of known strengthsolution of known strength
Testing methods - Direct Testing methods - Direct vs. Indirectvs. Indirect
IndirectIndirect– Treated glassTreated glass
Most common and oftenMost common and oftenbest methodbest method
– Treated organicsTreated organics Treating filter papers, cloth, woodTreating filter papers, cloth, wood May have May have adsorptionadsorption issues, especially issues, especially formulatedformulated
Treated Board StudiesTreated Board Studies
Holding patterns….Holding patterns….
How long to expose insects to How long to expose insects to insecticide?insecticide?– 1 hr, 4 hr, 24 hr?1 hr, 4 hr, 24 hr?– Irrelevant - pick one, dose will be Irrelevant - pick one, dose will be adjustedadjusted
When to count the dead?When to count the dead?– Post-treatment count?Post-treatment count?
OROR
– 24 or 48 hr after exposure?24 or 48 hr after exposure? Allows for kdr-resistant resistance detection.Allows for kdr-resistant resistance detection.
Resistance Profile vs. Resistance Profile vs. Diagnostic DoseDiagnostic Dose
Resistance ProfileResistance Profile– Profile requires increasing dosesProfile requires increasing doses
Usually Usually predatespredates use of Diagnostic Dose use of Diagnostic Dose
Single dose or Single dose or Diagnostic doseDiagnostic dose– Often used with formulated materialsOften used with formulated materials
Label Rate or a Given level exposureLabel Rate or a Given level exposure
Replication vs. PsuedoreplicationReplication vs. Psuedoreplication– All steps must be repeatedAll steps must be repeated
Includes weighing and mixing all solutionsIncludes weighing and mixing all solutions
0
20
40
60
80
100
% Survival
WyomingSchuylerSaratogaTioga
CS StrainStrain
0
20
40
60
80
100
% Survival
TiogaSaratogaSchuylerWyoming
Strain
1x3x10x30x100x
AnalysisAnalysis Control mortalityControl mortality
– CriticalCritical Many blood-feeding Diptera survive poorly Many blood-feeding Diptera survive poorly off hostoff host
– Often use “Abbott’s Formula”Often use “Abbott’s Formula”
Probit (logistic regression)Probit (logistic regression)– Generates mortality lineGenerates mortality line– Used for Lethal Dose (LD) calculationUsed for Lethal Dose (LD) calculation
Often used to identify 50% or 99% mortality Often used to identify 50% or 99% mortality valuevalue
(% Mortality - % Control (% Mortality - % Control Mortality) Mortality)
(1 - % Control (1 - % Control Mortality)Mortality)
= * 100
Real-world DataReal-world Data
House flies from 8 NY poultry and 4 House flies from 8 NY poultry and 4 dairy farms surveyeddairy farms surveyed
Many formulations testedMany formulations tested– Multiple active ingredientsMultiple active ingredients
Residual contact, feeding and larvicide Residual contact, feeding and larvicide assaysassays
Take homeTake home: Variations between sites, : Variations between sites, chemicals, methods, insecticide source….chemicals, methods, insecticide source….
Experimental CompoundExperimental Compound
HGFEDCBA0
20
40
60
80
100
% Survival
HGFEDCBA
County
1x3x10x30x100x
Methomyl (Feeding)Methomyl (Feeding)
A B C D E F G H0
20
40
60
80
100
% Survival
A B C D E F G H
County
1x3x10x30x100x
DimethoateDimethoate
A B C D E F G H0
20
40
60
80
100
% Survival
A B C D E F G H
County
1x3x10x30x100x
TetrachlorvinphosTetrachlorvinphos
A B C D E F G H0
20
40
60
80
100
% Survival
A B C D E F G H
County
1x3x10x30x100x
CyfluthrinCyfluthrin
A B C D E F G H0
20
40
60
80
100
% Survival
A B C D E F G HCounty
1x3x10x30x100x
PermethrinPermethrin
A B C D E F G H0
20
40
60
80
100
% Survival
A B C D E F G H
County
1x3x10x30x100x
PermethrinPermethrin
0
20
40
60
80
100
% Survival
WyomingSchuylerSaratogaTioga
CS StrainStrain
0
20
40
60
80
100
% Survival
TiogaSaratogaSchuylerWyoming
Strain
1x3x10x30x100x
TetrachlorvinphosTetrachlorvinphos
0
20
40
60
80
100
% Survival
WyomingSchuylerSaratogaTioga
CS StrainStrain
0
20
40
60
80
100
% Survival
TiogaSaratogaSchuylerWyoming
Strain
1x3x10x30x100x
DimethoateDimethoate
0
20
40
60
80
100
% Survival
WyomingSchuylerSaratogaTioga
CS StrainStrain
0
20
40
60
80
100
% Survival
TiogaSaratogaSchuylerWyoming
Strain
1x3x10x30x100x
Ectiban ECEctiban EC
0
20
40
60
80
100
% Survival
Flat Gloss Unpainted
CSTompkins
Schuyler
Panel Treatment
Strain
Ectiban WPEctiban WP
0
20
40
60
80
100
% Survival
Flat Gloss Unpainted
CSTompkins
Schuyler
Panel Treatment
Strain
Which one to use? Which one to use? Needs based.Needs based. To rapidly To rapidly identify identify resistanceresistance– Field collected****Field collected****– Lab rearedLab reared
PrecisionPrecision– Technical****Technical****– FormulatedFormulated
Deployed Deployed situationssituations– TechnicalTechnical– Formulated****Formulated****
Control failuresControl failures– TechnicalTechnical– Formulated****Formulated****
www.who.int/whopes/resistance/en/
WHO - Test KitsWHO - Test Kits
Purchasing KitsPurchasing Kits
Filth FliesFilth Flies