An example of arena choice bioassay using live workers

  Evaluation of the attractiveness of queen Dufour's gland pheromone extract on workers (Katzav-Gozansky et al., 2003).

  1. Apply the test material and also the solvent on two individually marked nurse bees in each assay.
  2. The amount of secretion applied onto a glass or live bee should be calculated relative to 1 queen equivalent.
    To illustrate, let’s assume that 1 queen equivalent (Qeq) of Dufour’s gland secretion is equal to 20mg (all constituents included). If the total ester content amounts to 14 µg, then for 1 Qeq of a blend of the esters, prepare a synthetic mixture of the gland esters in ethanol based on the relative proportion of each ester present in the total glandular extract (see Table 2) (Katzav-Gozansky et al., 1997).
  3. Apply the gland extract (2µl), its synthetic constituents (main esters) dissolved in ethanol using a micropipette. 
  4. Allow the solvent to evaporate for about 5 min.
  5. Colour-mark the treated bees on the thorax after applying the extracts on the abdomen and allow the paint to dry for another 5-10 mins.
  6. Introduce simultaneously extract and solvent treated bees into the arena.
  7. Observe the activity of the bees for 5 min and record the number of workers attracted to the test material (contacting the worker) at 30-sec time intervals (total of 10 times).

For each replicate the sum of contacts for treatment vs. control over 5 min is used as a measure of attraction. The level of preference of the treatment vs. control can be presented as the percentage of time intervals in which there is preference towards the treated bee. Statistical analyses can be performed by any commonly used program that performs non-parametric statistical tests such as Wilcoxon Signed Rank test, Kruskal-Wallis ANOVA or repeated measures ANOVA after appropriate transformation (see also the BEEBOOK paper on statistics by Pirk et al. (2013)).

Table 2. Ester composition of Dufour’s gland secretion of Apis mellifera queens and the amount used for preparation of ester synthetic blend (1 Qeq = 14 µg/ gland). *- Main compounds;  t- trace amount.


Amount of synthetic ester compound


 Tetradecyl dodecanoate


Tetradecyl (Z)-9-tetradecenoate t


Tetradecyl tetradecanoate*


Tetradecyl (Z)-9-hexadecenoatet  Tetradecyl (Z)-11-hexadecenoate*


Tetradecyl hexadecanoate

 Hexadecyl tetradecanoate*



Hexadecenyl hexadecenoate


Tetradecyl (Z)-9-octadecenoate*


Hexadecyl (Z)-9-hexadecenoate


Hexadecyl hexadecanoate


Octadecyl hexadecanoate