Agronomy Insects Dip Test Protocol to Determine Neonicotinoid Insecticide Resistance

Dip Test Protocol to Determine Neonicotinoid Insecticide Resistance

-

An important factor in managing insecticide resistance is to know the effectiveness of the product which is going to be applied. This can be accomplished by using dip tests, a simple technique in which adults or larvae are immersed for a few seconds in insecticide solutions. The mortality rate determined for each insecticide indicates the products which are likely to be most effective under field conditions.

Dip Test Items

Although dip test equipment is easy to obtain, it should be assembled well in advance of testing time. For this test you will need the following items:

  • A minimum of 30 Colorado potato beetles (CPB) (adults or larvae) per insecticide. In addition, you will need 30 or more CPB for the control (water test).
  • Commercial formulation of insecticides to be tested — Actara or Clutch.
  • Rubber gloves to keep any chemical from getting on your hands.
  • Plastic bottles (one litre or 35 oz each) with screw on tops to make up the insecticide solutions.
  • An eye dropper or a five millilitre plastic syringe to measure the volume of insecticide which is going to be mixed with water. Eye droppers and syringes are available in the pharmacy section of most drug stores.
  • Plastic containers approximately 250 ml (8 oz) each, to pour dip solutions.
  • A 7.5 cm (3 inch) tea strainer to hold the beetles or larvae as they are being dipped.
  • Styrofoam/paper coffee cups with perforated lids to keep the beetles in after the dip test is completed.
  • Absorbent paper towels.
  • A permanent type marking pen.
  • Record sheet.

Collection of Beetles or Larvae

Collect beetles or larvae from several places in the field, especially from problem areas. Usually, the life stage selected for dip testing is the one which is causing the greatest defoliation. Place the beetles or larvae in a plastic container with a perforated lid to allow air movement. Keep the container out of direct sunlight. If possible, collect the insects the same day on which the dip test will be done. However, if the dip test is going to be conducted 24 hours after the beetles or larvae were gathered, place them in the refrigerator.

All the chemical insecticides, either dilute or full strength, can be toxic when spilled on your hands. Wear personal protective equipment (PPE), rubber gloves and face mask, throughout the test.

Making up the insecticide solution: The concentration of the dipping solution of Actara or Clutch must be the same as which is used for foliar sprays per acre based on a spray volume of 100 L per acre (check the label to obtain the correct foliar spray rate). Make sure that the plastic syringes/eye dropper you use to make up the stock solutions are clean and dry.

Dipping the Beetles or Larvae

Pour the insecticide solution into the plastic container being used for dipping. Place either 30 beetles or larvae in the tea strainer. Place the tea strainer in the insecticide solution and swish the CPB around for approximately five seconds to make sure all beetles or larvae are covered with the solution. At the end of the five seconds, lift the strainer, let it drain for a moment and place it on absorbent paper towels for about half a minute to take up any free solution. Dump the treated beetles into a labelled Styrofoam/paper coffee cup. Put the perforated lid on.

It is recommended to do the control test (dipping CPB in water) first and also to wash the tea strainer thoroughly between dip tests of different insecticides.

Immediately after you have finished with the dip test, place the cups in an area that is close to 20 C and out of direct sunlight.

Determination of Mortality

Check the beetles after 12 to 18 hours. Remove them from the cup and place them on a piece of paper to determine mortality. The beetles sometimes play dead. If the legs are held tightly to the body, the beetle is probably not dead. If the area is warm or directly illuminated with a light bulb, the beetles or larvae which survived the test start to move and walk.

Beetles which are lying on their backs and have extended legs, sunken abdomens and cannot walk without falling over should be considered dead.

If between 85 per cent to 100 per cent (25 or more out of 30) CPB are dead in an insecticide test, that insecticide will give reasonably good control in the field.

If between 75 per cent to 84 per cent (22 to 24 out of 30) CPB are dead, most of the population is still susceptible, but resistance should develop quickly if that insecticide is applied more than once during the season.

If 50 per cent or less (15 or less out of 30) CPB are dead and you use that insecticide, it may kill only some CPB or it may be completely ineffective.

Important! The water treatment or control indicates the accuracy of the dip test. If more than 10 per cent (3 out of 30 beetles or larvae) in the control test are dead, you should repeat the test with healthier CPB. (One of the reasons for mortality in the control test could be keeping the CPB at high temperatures in a closed container after gathering them in the field.)

Labelling the Containers

The containers where the insecticide solutions (stock solutions) are made up should be labelled as follows:

  • Insecticide trade name and formulation Concentration of dipping solution (e.g.4. 5 ml/10 L)
  • Date of preparation:
  • The styrofoam/paper coffee cups where the beetles are placed after the test should indicate:
    • Insecticide trade name
    • Location (field where the beetles came from)
    • Date of the test

Keeping Records

Dip test records can be kept on a record sheet which should include the following information:

  • Date
  • Field
  • Insecticide
  • Concentration of stock solution (e.g 4.5 mL in 10L water)
  • Number of CPB tested vs dead = percentage of mortality
  • Adults
  • Large larvae

It is better to test large larvae than small larvae. Small larvae are more susceptible to insecticides.