Methods in Anopheles Research - MR4

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Chapter 5 : Insecticide Resistance5.1 Insecticide Resistance Bioassays5.1.3 Guidelines for Evaluating Insecticide Resistance in Vectors using the CDC Bottle BioassayPage 9 of 24Figure 5.1.3.3: Coating the bottom of the bottleFigure 5.1.3.4: Coating the top of the bottleFigure 5.1.3.5: Coating the sides of the bottleFigure 5.1.3.6: Removing caps and rolling thebottles on their sidesCDC bottle bioassay methodGeneral considerations1. Use a filter in the aspirator to avoid inhaling mosquitoes or insect fragments;2. Blow gently to expel the mosquitoes into the bottles. If you blow too hard, the mosquitoes can bedamaged by hitting the sides of the bottle and killed before the insecticide has a chance to do so;3. Be careful not to touch the inside of the bottle with the aspirator, as this may contaminate theaspirator;4. Remember that the number of mosquitoes in the each of the test bottles does not need to beequal;5. Determine species composition of mosquitoes either before or after the bioassay is conducted(Box 2).
Chapter 5 : Insecticide Resistance5.1 Insecticide Resistance Bioassays5.1.3 Guidelines for Evaluating Insecticide Resistance in Vectors using the CDC Bottle BioassayPage 10 of 24Bioassay procedureThe bioassay can be performed with the bottles in an upright position or with the bottles lying on theirsides. The important thing is to be consistent and follow the same procedure each time.The steps:1. Using an aspirator, introduce 10–25 mosquitoes into the control bottle. It is not necessary tocount the mosquitoes; the exact number does not matter;2. Introduce 10–25 mosquitoes into each test bottle; again, the exact number does not matter(Figure 5.1.3.7);3. Start a timer. Be sure to examine the bottles at Time 0 and count the number of dead and/or livemosquitoes;4. If you find dead mosquitoes at Time 0, make a note of them on the form (Appendix 3);5. Record how many mosquitoes are dead or alive, whichever is easier to count, every 15 minutesuntil all are dead, or up to 2 hours (Figure 5.1.3.8). It is not necessary to continue the bioassaybeyond 2 hours;6. Record these data on the reporting form (Appendix 3);7. Graph the total percent mortality (Y axis) against time (X axis) for all replicates consideredtogether using a linear scale;8. Remember that mortality at diagnostic time is the most critical value because it represents thethreshold between susceptibility and resistance. Refer to Table 5.1.3.1 for diagnostic doses andtimes for commonly used insecticides;9. Take into consideration mortality in the control bottle at 2 hours (end of the bioassay) whenreporting the results of the bioassay (See validity of the data section below). Use Abbot’s formulato correct results if the mortality at 2 hours in the control bottle is between 3% and 10%. You mayneed to discard the bioassay results if mortality in the control bottle at the end of the test was>10%.Mosquitoes are considered dead if they can no longer stand. See Box 3 for more information.A timer could be started for each bottle, but it is sufficient to start one timer when the first or last bottlereceives its mosquitoes. It is, however, important to be consistent and follow the same timer startprocedure each time. Mosquitoes alive at the diagnostic time (Table 5.1.3.1) represent mosquitoesresistant to the insecticide being tested. These mosquitoes may be transferred to a sleeved carton forfurther analysis (e.g., molecular or biochemical assays). Mosquitoes flying at the end of the bioassay inthe control bottle may need to be killed to get an accurate count. Mosquitoes can be killed by freezing orstunning them.Box 3: Notes about mortality criteria. “Dead” mosquitoes are mosquitoes that cannot stand. It helps to gently rotate the bottle while taking the count. Immobile mosquitoes that slide along the curvature of the bottle can be easily categorizedas dead. It is easier to count the number of dead mosquitoes in the first readings of the bioassay, andit is easier to count the number of live mosquitoes when few remain alive. In the end, the percentage of dead mosquitoes at the diagnostic time (dead mosquitoes/total of mosquitoes in the assay) is the most important value in the graph.
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Preface to Second EditionWhen Mark
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Chapter 1 : Insectary Operation1.1
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