Bioassays for contact substances

1. A stainless steel ring (56 mm inner Æ, 2–3 mm height) and 2 glass circles (62 mm diameter; Na-Ca glass) are cleaned with acetone and hexane or pentane to form the testing arena.

2. Apply the product to be tested and the control solution on the arena pieces.

Various concentrations of the products are tested. See the BEEBOOK paper on methods for toxicological studies (Medrzycki et al., 2013) to define these concentrations.

The application of the active ingredient on the arena pieces varies according to its physico-chemical properties.

2.1. for water soluble active ingredients (polar compound):

   2.1.1. Mix the active ingredient with a convenient solvent.

   2.1.2. Spray the glass disks and ring with a solution of the compound as evenly as possible.

This can be done by means of a “Potter precision spray tower” (e.g. Burkard Manufacturing Co; UK) (Milani, 2001). To do so, the reservoir is loaded with 1 ml of solution; the distance of the sprayed surface from the bottom end of the tube is set at 11 mm and a nozzle 0.0275 inches is used. The pressure is adjusted (usually in the range 350–500 hPa) until the amount of solution deposited is 1 ± 0.05 mg/cm2. Alternatively, if such piece of equipment is not available a glass Petri dish can be used as arena. The solution (active ingredient in a solvent of low boiling point) is poured in the dish so as to cover the whole bottom of the dish and left to evaporate under a fume hood. Depending on the surface tension of the active ingredient, this will result in an uniform layer of substance at the bottom of the dish. Varroa mites can thus be exposed to the substance in the Petri dish. This method cannot be used when the surface tension of the ingredient is too high and droplets are formed on the Petri dish.


2.2. For lipid soluble (apolar compound) active ingredients:

   2.2.1. Melt 10 g of paraffin wax (e.g. Merck 7151, melting point 46-48 °C) in a glass container kept in a water bath at 60 °C.

   2.2.2. Dissolve the required amount of the active ingredient in a convenient solvent (e.g. hexane or acetone).

   2.2.3. Add this solution to the melted wax.

   The solvent alone is added to the control wax.

   2.2.4. Weigh glass disks and iron rings before coating it with the wax.

   2.2.5. Stir the mixture for 30 min.

   2.2.6. Immerse the steel rings in the molten paraffin wax, one side of the glass disks is coated by lowering the disks onto the molten paraffin.

   2.2.7. Weigh glass disks and iron rings after coating.

   Discard the arenas (ring + glass circles) with a total amount of coating outside the range 1.6-2.0 g.

   2.2.8. Keep the arena pieces for at least 24 h at room temperature to allow for the solvent to evaporate.

   2.2.9. Store at 32.5 °C until they are used.

3. Place the ring between the glass circles so as to build a cage.

The cages are used within 60 h of preparation, for not more than three assays.

4. Introduce 10 to 15 varroa mites in this cage and bind the pieces together with droplets of melted wax.

Mites collected from spinning larvae, stretched larvae, white eyed pupae and dark eyed with white and pale body are used. See the section ‘Obtaining brood and adults of known age’ estimating pupa age of the BEEBOOK paper on miscellaneous methods (Human et al., 2013) for determining the age of pupae.

5. After 4 h transfer mites into a clean glass Petri dish (60 mm Æ) with two or three worker larvae taken from cells 0–24 h after capping (obtained as described in section 3.1.4. ‘Collecting mites from the brood’) or with two or three white eye pupae (4-5 days after capping).

6. Observe the mites under a dissecting microscope, 4 (i.e. at the time of transfer into the Petri dish), 24 and 48 h after the beginning of the treatment and classify as:

   6.1. Mobile: they walk around when on their legs, non-stimulated or after stimulation.

   6.2. Paralysed: they move one or more appendages, non-stimulated or after stimulation, but they cannot move around.

   6.3. Dead: immobile and do not react to 3 subsequent stimulations.

A clean tooth pick or needle can be used to stimulate the mites by touching their legs. New tooth picks or cleaned needles should be used for stimulating control groups to avoid their contamination with residues of active ingredients from treated mites. The assays are carried out at 32.5 °C and 60-70 % RH. If the mortality in the controls exceeds 30 %, the replicate is excluded. Each experiment is replicated with a sufficient number of series of cages. To determine the sample size, refer to the BEEBOOK paper on methods for toxicological studies (Medrzycki et al., 2013) and the BEEBOOK paper on statistics (Pirk et al., 2013). If mites are scarce, more replications are carried out and more mites are assayed at doses around the median lethal density, to increase statistical resolution in this region.