5.4.3. Arena tests

A disposable plastic Petri dish (15 x 2 cm) or 9 cm in diameter lined with a filter paper, can be used as a bioassay arena (Kaminsky et al., 1990; Wossler and Crewe, 1999). The system is especially suitable for a two choice bioasay. For stimulus introduction, side slits for insertion of the tested material or openings for introduction of bees on both sides of the cover can be easily prepared (Katzav-Gozansky et al., 2003). Individual behaviour of 10-20 worker bees can be observed at a time in such an arena. The best approach is  to use workers aged 1-12 days which  normally attend to the queen (Seeley, 1982). If the exact age of the bees is unknown, workers can be collected from the open brood area of a queenright colony and used for the screening bioassay. Freshly collected bees are best for this study. However, if it is intented  to test the responsiveness of workers from a special age and task, these need to be prepared in advance by marking bees at emergence  (see the section on obtaining brood and workers of known age in the BEEBOOK paper on miscellaneous methods (Human et al., 2013)).

  1. 1.Introduce the glass slides (treatment and control) or marked workers (Fig. 7) simultaneously to the arena through the side slits.
  2. Place the arena in a temperature controlled room (25-27°C). Either red or day light will do for this assay.
  3. Record behaviour towards each one at pre-determined time intervals.

Since  the volatility of the tested component is expected to affect the active space of the chemical and thus the detection time, the length of the observation need to be determined in preliminary studies.

This set up enables simultaneous evaluation of multiple arenas.

Pros: It is fast and highly reproducible. It is also a sensitive bioassay. Especially well suited for evaluation of chemicals of low volatility. It requires minimal equipment investment. Positive response can be obtained with low amount of material i.e. 10-7 Qeq of synthetic 4 component queen mandibular pheromone (Kaminski et al., 1990) and 1/3 Qeq of Dufours gland extract (Katzav-Gozansky et al., 2003). This bioassay enables the testing of worker responses based on their specific ages and tasks.

Cons: This type of assay examines honey bee behaviour out of the natural context; the assay conducted in closed atmosphere with mostly still air can be problematic for evaluation of highly volatile compounds as the arenas could become saturated with volatiles rather quickly, thus eliminating the gradient to which the test organism could respond (Hare, 1998).

Fig. 7. The arena bioassay in the Petri dish. The red marked bee is pheromone treated while the yellow one is solvent treated control. The blue marks indicate openings for bees' introduction. Arrows indicate slots,  for the glass slide introduction, when the latter are used as the surrogates.

figure7 An example of arena choice bioassay using live workers