Introduction

This method, validated by Dr Thieme (BTL Bio-Test Labor GmbH Sagerheide), is an adaptation of the vial test method (IRAC #11) currently used for monitoring sensitivity of Meligethes aeneus populations to pyrethroids.

Method

1. Collect approximately 300 adult beetles at different locations across the infested field. Store beetles in an aerated plastic container. Place some dry paper towel at the bottom of the container, and add some oilseed rape leaves plus two or three inflorescences as food source (Figures 1a, 1b). The insects should not be subjected to excessive temperature, humidity or starvation stress after collection.

2. Use the attached recording sheet for sampling details and other information that may be useful for tracking samples and interpreting susceptibility results later on.

3. Ship the containers as quickly as possible to the test laboratory; transportation method should avoid excessive temperature, humidity or starvation stress.

4. It is recommended that on arrival to the laboratory, the beetles be released into a ventilated holding cage and left to recover overnight.

5. The standard test oxadiazine is formulated indoxacarb. No other active substance from this chemical family has been tested so far.

6. The test containers are glass vials with an inner surface area of 20-40 cm2. Newly purchased vials should be cleaned of potential residues from their manufacture by soaking overnight in soapy water, rinsing with acetone and air drying for at least 4 hours before use.

7. Determine the surface area of the glass vials by:

   • h = height of the vial
   • r = radius of the bottom
   • Surface Area = area of bottom + area of the side
   • Surface Area = π r 2 + (2 πr)* h

   

8. Prepare accurate dilutions of the formulated compound with 5% water + 95% acetone. In order to generate accurate estimates of the susceptibility of pollen beetles, a set of five to six concentrations of indoxacarb should be tested. The suitable test concentrations in ng ai per cm2 inner glass surface have been determined as follows:

   • 255 ng/cm² (100% of the European field application rate of 25.5 g ai/ha),
   • 90.5 ng/cm²
   • 63.75 ng/cm²
   • 31.88 ng/cm²
   • 9.4 ng/cm²
   • 1.99 ng/cm²
   • Water + acetone only as Control

   If it is not possible to test the full range of concentrations, 2 rates + the untreated control are then recommended as follows:
   255 ng/cm² (100% of the European field application rate of 25.5 g ai/ha),
   63.75 ng/cm² (25% of the European field application rate of 25.5 g ai/ha).
   Water + acetone only as Control

9. Glass vials should be filled with 500-1500 μl (depending on vial size) of solution and rotated at room temperature until the water/acetone mixture is completely evaporated, at least for 4 hours.

10. Treated vials can be kept for 28 days after production, stored in a fridge (5 ± 2°C), or for 14 days stored at room temperature (20 ± 2°C).

11. A minimum of three replicates of each concentration and control are required.

12. Place a minimum of ten adult beetles per vial (a funnel can be helpful in transferring the beetles to the vial). It is recommended to avoid any hand contact with the beetles during the transfer. Transferring insects in a cool environment (cold chamber at 10°C) will slow down movement of the insects and may facilitate this operation. Then cap and store the vials upright at 20 ± 2ºC in a dark ct-room at 20 ± 2°C.

13. After twenty four hours, count affected (including dead and moribund) and alive beetles. Beetles, which cannot make co-ordinated movement over a period of 60 seconds, should be considered dead or affected.

14. Express results as percentage mortalities. If control mortality is greater than 20% the study should be considered as invalid for the purposes of resistance monitoring.

15. No case of resistance or clear reduction of pollen beetle susceptibility has been detected since the beginning of susceptibility monitoring with indoxacarb, though it is not possible to propose a ‘susceptibility rating scheme’ at this stage. The pdf file of the method (link above) shows a few examples of results that should be expected.

Precautions & Notes

1. Where glass equipment is used it must be adequately cleaned with an appropriate organic solvent before re-use to prevent cross-contamination.

2. Two different formulations of indoxacarb have been tested so far: a solid WG formulation containing 30% indoxacarb and a liquid EC formulation containing 150 g/L of indoxacarb. Tested side by side, there was no significant difference in the response provided by these 2 formulations, which means both of them could be used for the evaluation of pollen beetle’s susceptibility to indoxacarb.

3. The doses indicated for testing are expressed in ng/cm² of active substance.

Comments

Sample recording and assessment sheets can be found in the pdf file of the method.