PROCEDURE FOR CALCULATING TIME-MORTALITY CURVES BY THE BOTTLE BIOASSAY

PROCEDURE FOR CALCULATING TIME-MORTALITY CURVES BY THE BOTTLE BIOASSAY

 

By Jack Petersen

 

Introduction.

 

Insecticide resistance is a threat to the success of any mosquito control program.  Early detection and proper management decisions are essential to keeping this problem in check.  One method of determining the response of adult mosquitoes to a given pesticide is the bottle bioassay.

 

Brief Overview.

 

A standard dose is used to discriminate between insects susceptible to an insecticide and those that are resistant.  The bottle bioassay is used as an indicator of the insect’s response to that dose, specifically, how many minutes it takes to kill all the test mosquitoes.

 

Materials Needed

 

250 ml Wheaton bottles

Pipettes, glass, 1 ml calibrated in hundreths

A.C.S. Acetone

Chemical(s) to be tested

Aspirator

Timer

Mosquitoes

 

Getting Started.

  1. Start with clean, dry bottles.  If possible, heat bottles to 350 degrees F in an oven for 15-20 min. before use.  Allow the bottles to return to room temperature.
  2. Prepare the stock solutions.  If stock solutions are already made and have been refrigerated, make sure you allow enough time for them to come to room temperature before use.
  3. Formulations of insecticides are given as appendices in another part of this procedural guide.  Once the stock solution is prepared, it can be stored in the refrigerator in light-proof bottles until needed.
  4. Mark your bottles.  Make sure you mark the cap as well so you can keep individual bottles and their caps together.  This is important because you will be coating the entire bottle, including the inside of the cap.  To ensure the bottle has the proper dosage you must keep the cap with its respective bottle.

Preparing the Bottle.

 

  1. Pipette into the bottle 1 ml of A.C.S. acetone plus the active ingredient (A.I.) to be tested.  The precise amount of A. I. to be added to each bottle depends on the chemical.  See Table 1.
  2. Also prepare one or more control bottles that contain 1 ml of acetone, but no active ingredient.  These bottles are an important test of proper preparation.
  3. Make sure the bottle cap is secure so that no acetone evaporates until you are ready for it to do so.
  4. Swirl the bottle gently so that the acetone evenly coats the bottom.
  5. Continue turning the bottle while inclining the bottle so that the sides are evenly coated.  Do this slowly and carefully, looking for streaks.  The interior of the bottle must be evenly coated.
  6. Incline the bottle so that the acetone puddles in the “shoulder.”  Rotate the bottle several times to distribute the acetone evenly.
  7. Turn the bottle completely up side down.  Did any acetone escape?  If it did, reject that bottle and start over again with another bottle.
  8. Coat the inside of the cap.
  9. Turn the bottle on its side and lay it on a flat surface.
  10. Gently vent the bottle by unscrewing the cap.  You should hear an audible “pssst!”
  11. Now roll the bottle to keep the insecticide evenly distributed on the walls while the acetone evaporates.
  12. Follow this procedure with all the bottles until no liquid acetone is visible.
  13. Allow the bottles to dry with lids off for at least two (2) hours before proceeding.
  14. Store the bottles in a dark, cool, dry place

 

The Bioassay.

 

  1. Aspirate 10-15 mosquitoes into each bottle.  Use a number you can easily count with accuracy as the mosquitoes fly about.  It works best to collect all of the mosquitoes for one bottle in a mouth aspirator and introduce them all at once with a gentle puff of air.  Try to avoid adding excessive moisture from your breath.  A mechanical aspirator works best.
  2. Examine the bottle to be sure all mosquitoes survived the transfer process.  If you find any dead mosquitoes, record the number.  You will need to substract this number from each timed reading.
  3. Start a timer and record every 15 minutes how many mosquitoes are dead or alive (whichever is easier to count).  Continue until all mosquitoes are dead.  In some cases, when mosquitoes are resistant to the pesticide, it may be necessary to set an arbitrary maximum time limit such as five hours.
  4. Record your counts on the data sheet that accompanies this procedure.
  5. Continue until all mosquitoes are dead or a pre-determined time has expired.
  6. Count the total number of mosquitoes in each bottle and calculate the percent mortality for each 15 minute interval.
  7. Plot the percent mortality (y axis) against time (x axis) using a logarithmic scale for the percent mortality.
  8. Compare the test mosquitoes with baseline date established for susceptible mosquitoes of the same species.

 

Clean up.

 

  1. When you are finished with your bottles or the pesticide is too degraded to use any longer, triple rinse them with acetone, wash them with warm soapy water and rinse.  Place them in an oven to thoroughly dry before using them again.
  2. If you are uncertain whether the bottles are completely clean, introduce some susceptible mosquitoes into the bottles after you dry them.  The mosquitoes should not die in three hours or less.  If they do, clean the bottles again.

 

Table 1.  Recommended bottle dosages.

 

Active Ingredient

Final concentration/bottle

Malathion

474 g/bottle

Naled

25 g/bottle

Permethrin

43 g/bottle

Pyrethrin

30 g/bottle

Resmethrin

30 g/bottle

d-Phenothrin

22 g/bottle

DEF

125 g/bottle

TPP

125 g/bottle

PBO

400 g/bottle

 

Revised:  25 January 2002;  Revised:  26 April 2002;  Revised 10 February 2003;  Revised 12 November 2004;   Revised 25 May 2007; :Revised 9 March 2012

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