4.5.3.2. Mite reproduction

In the case of the compounds affecting mite reproduction, field testing involves treating brood cells with the chemical under study and evaluating both the fertility and fecundity of the mites reproducing in the cell.

 

1. Chose combs containing brood close to being capped.

2. Mark all the capped cells on a transparent sheet placed over the comb.

3. Replace the comb in the colony for two hours for workers to carry on capping cells.

4. Bring the combs to the laboratory after the two hours.

6. Dissolve the compound in an appropriate solvent.

The dose used for the field bioassay is normally the most active in the laboratory bioassay.

6. Treat groups of freshly capped (unmarked) worker cells by injecting 1 µl of the solution with a 10 µl Hamilton syringe under the capping.

Do not insert the syringe too deep into the cell to avoid hurting the larva. Beware that the solvent could dissolve the wax of the cell walls.

7. Treat an equal number of cells with 1 µl of the solvent as a control.

8. Choose groups of control and treated cells on both side of the comb, separated by at least one cell, which is left untouched to avoid contaminations.

9. Mark the position of the control and experimental cells on a transparent sheet placed over the comb.

10. Return the combs to the hive within 3 h.

11. Bring the comb to the laboratory 11 days later, when the bees are about to emerge.

12. Identify treated and control cells using the transparent sheet.

13. Count, uncap and inspect intact cells.

14. Note the condition of the infested pupae.

15. Collect mother mites and their offspring.

16. Mount on microscope slides and identify developmental stages as described in section 4.3.3. ‘How to measure reproductive success’.

In particular, the number of offspring and the number of mated daughters (i.e., the number of adult daughters in cells containing an adult male), are considered.

The effects of the solvent on the reproduction of V. destructor are studied by comparing the reproduction of mites in cells injected with 1 µl solvent and in sham-treated infested cells (syringe was introduced, but no solvent was injected). Proportions of reproducing mites out of the total mites found in cells are compared using G-tests (with the Williams’ correction). The number of offspring and that of mated daughters per mother mite in treated and control groups can be compared using a two-sample randomisation test. The randomization distribution should be resampled an adapted number of times (e.g. 106 times).