18.104.22.168. Mites used in susceptibility bioassays
For this assay, mites that would be exposed to the product in colonies in the apiaries should be used. Some products affect only phoretic mites, others also affect mites in brood cells. Adult mites at the reproductive stage might have different susceptibility compared to phoretic mites or to their offspring because their cuticle is not hardened or because of physiological differences. Milani (1995) and Milani and Della Vedova (1996) tested compounds only affecting phoretic mites, but showed that mortality was more homogeneous in mites collected from the brood. This might be due to a more homogeneous physiological status compared to phoretic mites. It is therefore recommended to test all life stages to obtain a complete picture of mite susceptibility.
When brood mites are tested, they are collected from combs (or pieces of comb) of infested colonies after opening and inspection of capped cells. Mites parasitizing brood of different developmental stage have different susceptibilities to acaricides (Milani and Della Vedova, 1996). For the tests, they are therefore grouped according to the age of their brood host and assayed separately. The age of larvae or pupae inhabiting these cells can be pre-determined by marking at the capping stage and opening it at a given time. Alternatively, the approximate age of the brood can be inferred on the basis of the morphology and pigmentation of the larva or the pupa (see section ‘Obtaining brood and adults of known age’ in the BEEBOOK paper on miscellaneous methods (Human et al., 2013). Varroa mites from different brood stages can be pooled if previous results indicate no differences among development stages (Milani, 1995). Differences between mite developmental stages might also influence their susceptibility to active ingredients, independently of host development, but this has not been shown yet.
Mites are kept on their host larva or pupa in glass Petri dishes until a sufficient number is collected. This ensures they can feed if hungry and the availability of their own host ensures that their physiological status is not changed. Since mites might stray from their host larva or pupa and climb onto another, only hosts at the same development stage should be kept in any given dish.