General experimental establishment

  1. Establish equalized 3-frame observation hives (see the respective BEEBOOK paper Delaplane et al., 2013) according to the parameters set for the study (one brood frame and two honey frames; queenright or queenless, etc., see the BEEBOOK paper on behavioural methods by Scheiner et al., 2013 for details on establishing and maintaining observation hives) and remove any beetles currently living in the colony using aspirators (see Fig. 6).
  2. The observation hives should be kept in a dark room, with no outside or artificial light shining on the hive.
  3. According to the needs of the experiment, collect adult SHBs from rearing programs (see section 3.1.2.) or from infested field colonies. The beetles can be all males, all females, age-cohort specific, etc. per the needs of the experiment. Adult beetles should be sexed if required (see section 3.1.3.) and introduced into the observation hives after dusk, the preferred time window for SHB flights (Neumann et al., 2012).
  4. Hives should be monitored at least twice daily (Neumann et al., 2001c; Ellis et al. 2003c,d, 2004a,b) at time windows suitable for the experimental needs (morning and afternoon or only after local dusk for nocturnal behaviour). All night observations of the hives should be done using red lights to minimize behaviour disturbance to colonies. It may be necessary to use a small flashlight to find beetles confined in particularly hard-to-view areas. The order of observed hives should be altered at a daily basis at each time window.
  5. During behavioural studies, it is essential to precisely define the behavioural categories/ pattern. They have to be clearly defined and distinct; under no circumstance should the observer come into the situation that a certain observed behavioural pattern could be either behaviour 1 or behaviour 2. As a hypothetical example let’s assume that two categories are labelled “walking” and “running”. These categories would be examples of poorly defined behaviours, since they do not give a clear objective definition. The categories “walking, speed less than 5mm/sec” and “running, speed greater than 5mm/sec” would be clearly defined and are distinct. As another hypothetical example, the categories “feeding” and “trophallactic interactions” would be not helpful category choices since they overlap.
  6. In most behavioural studies, it is important to know where the interaction took place, especially in a social insect colony like a honey bee colony and especially in the case of SHB/ honey bee interactions. Therefore, it is useful to superimpose a transparent grid of squares onto both side windows of an observation hive to fix the location of the interaction. Obviously, the size of the square determines the spatial resolution. The general consensus is to use 5 x 5 cm squares (Moritz et al., 2001; Neumann et al., 2001b, 2003; Ellis et al., 2003d), which then could be even further subdivided with slightly drawn internal 1 x 1 cm squares (Ellis et al., 2003d). One has to avoid overloading the side windows with markings to ensure that one can actually observe the behaviour. The labelling of the rows and columns should be included on the edge of the grid. To avoid confusion, the rows should be labelled with numbers and the columns with letters (or vice versa). It is highly recommended when preforming observations on both sides of an observation frame to continuously label the columns around both sides. For example with a 6 x 9 grid on each side, the rows are labelled 1-9 and the columns are labelled A-F on one side and G-L on the opposite side.
  7. At each observation period, the observer should screen the colony in a left-to-right pattern, following the uppermost 5 × 5 cm bold square. This pattern should be followed with the second row of 5 × 5 cm bold squares, continuing to the bottom of the grid. This observation pattern minimizes the chance that the same area will be viewed more than one time and that behaviour will be double counted. Both sides of the observation hive should be monitored this way.