Methods in Anopheles Research - MR4

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Chapter 5 : Insecticide Resistance Monitoring5.1 Insecticide Resistance Bioassays5.1.1 Larval Insecticide Resistance AssaysPage 1 of 4Chapter 5 : Insecticide Resistance Monitoring5.1 Insecticide Resistance Bioassays5.1.1 Larval Insecticide Resistance AssaysMR4 StaffIntroductionInsecticide-susceptibility defined stocks require routine quality control measures to determine whether acontamination event has occurred. Exposure is also conducted to ensure that the level of resistance hasnot changed. Larval exposures are simple to conduct since they are a non-flying stage and exposurelevels are consistent in the aquatic environment. There are numerous variations for larval exposures, butthe MR4 uses fixed time and dose treatments of L4s routinely because of their ease. Exposure of otherstages and even embryos is also possible using similar methods.In each of these assays, L4s are subjected to a known concentration of an insecticide for a fixed timeperiod (French and Kitzmiller 1963). Because some mortality may occur even among resistant individuals,if the purpose is selection for a resistant colony, a large cohort should be tested to perpetuate the colony.Experience with a particular insecticide will indicate how long the exposure should be and what timeperiods are appropriate for your application. The exact time and concentration necessary for causingmortality or boosting insecticide resistance is best determined utilizing dose-response curves.Some typical discriminating concentrations and treatment times for L4s (solvent).• DDT: 0.4ppm DDT (ethanol) for 24 hours.• Permethrin: 1ppm permethrin (ethanol) for 24 hours.• Propoxur: 20ppm propoxur (acetone) for 1 hour.• Dieldrin: 1ppm dieldrin (ethanol) for 1 hour.• Malathion: 1ppm malathion (ethanol) for 24 hours.Materials 1• Concentrated insecticide from which stock dilution will be prepared (1000 X the final treatmentconcentration is recommended)• Glass graduated cylinders• Volumetric flask1Many of the insecticides will be formulated in solvents that dissolve plastics - especially acetone - soglass bottles and pipettes are recommended.
Chapter 5 : Insecticide Resistance Monitoring5.1 Insecticide Resistance Bioassays5.1.1 Larval Insecticide Resistance AssaysPage 2 of 4• Glass pipettes• Pipetting device• 100 ml glass bottles with screw tops for insecticide stock storage• Dedicated treatment containers. These can be assigned to a particular insecticide, or if they aremade of glass or metal, they can be cleaned thoroughly with solvent for reuse. Polypropylenefood cups and other disposable containers are good for treatments. Treatment containers shouldnot be mixed with colony maintenance trays.• Solvents for dilution of insecticide (see above)• Stainless steel or nylon strainer fine enough to collect L4s.ProtocolExposure:1. Prepare the stock solution of insecticide. 22. Prepare the insecticide dilution in water at the desired concentration.3. Collect early stage L4s ensuring that sufficient numbers for a backup are retained in the event of anunexpected result or accident. For example, MQB once accidentally washed resistant larvae withacetone! Resistance was not observed. Do not expose pharate pupae. They are often more resistantthan L4s. 34. Drain water off of larvae using a fine mesh. Stainless steel kitchen handheld strainers or improviseddevices are suitable.5. Transfer the larvae to the empty treatment pans by tapping the screen containing larvae sharply onthe side of the treatment tray or by washing them in. Attempt to add as little water with the larvae tothe pans as possible (Figure 5.1.1.1) to avoid insecticide dilution. Aspirate out excess water.6. Add insecticide to the pans using a volume similar to that used for larval culture (~ 1 ml / L4) (Figure5.1.1.2). After a few minutes, susceptible larvae can often be recognized by unusual twitchingmovement. Typically, these larvae will not recover and will be classified as moribund (French andKitzmiller 1963).7. For all assays that are longer than 1 hour, feed larvae to ensure mortality is due to the insecticide andnot starvation.8. At the end of the exposure, strain off the larvae and wash thoroughly with water before returning to aculture tray.2 For routine use of insecticides at the same concentration, we have found it convenient to formulateinsecticides at 1000 X the concentration desired. This makes formulation of the final dilution simple (1 mlper liter) and keeps the solvent concentration sufficiently low that it alone does not cause mortality.3 A susceptible reference stock is often necessary to ensure that a lethal dose has been delivered. Forroutine authentication purposes, this may be a stock of a different species.
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\Methods inAnopheles ResearchSecond
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Preface to Second EditionWhen Mark
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Chapter 1 : Insectary Operation1.1
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Chapter 9 : Obtaining Live Material
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Chapter 9 : Obtaining Live Material
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