6.2.1. Example protocols

The most popular and versatile apparatus for studying aversive conditioning is the shuttle box (also known as a choice chamber). A shuttle box (Fig. 6) is a two-compartment chamber between which an unrestrained bee moves back and forth (“shuttles”). The shuttle box can accommodate all types of aversive conditioning designs in the areas of escape, punishment, and avoidance (see Table 2). Molecular studies of learning in the honey bee can also be performed in a shuttle box (Agarwal et al., 2011). If a researcher is interested in the effect of reward on learning, escape conditioning is a good alternative to an appetitive reward. To perform escape conditioning:

1. Select the parameters for the training variables. Table 3 provides a list.

2. Place the honey bee in the shuttle box for a 5 minute adaptation period.

3. Present an electric shock to the bee while it stays in one compartment. As a response to this shock, the bee will escape from the shock and enter a different compartment. The shock-free time in this compartment constitutes the reward.

4. Measure the time the bee takes to terminate (escape) the shock.

Escape conditioning is an especially useful aversive conditioning technique for bee researchers, because it is simple to use and there is no concern for motivational issues associated with food rewards in situations where the bee is confined and unable to unload. In addition, the results of escape conditioning are similar to those found with food rewards (see Macintosh, 1974 and Campbell and Church, 1969).

The shuttle box can also be used to study punishment, another simple type of aversive conditioning. Here, the honey bee receives an aversive stimulus, such as shock, as a consequence of entering a compartment. As a result of this contingency, the bee decreases the probability of shuttling. This situation is ideal for bee researchers looking for a situation that produces the opposite effect of reward training. If a researcher is looking for a complex conditioning situation, one protocol to use with a shuttle box is signalled avoidance. Signalled avoidance has the interesting property that it contains elements of both Pavlovian and instrumental conditioning. The experiment begins with a conditioned stimulus (CS) predicting an impending aversive event such as electric shock. If the honey bee does not respond to the CS by entering the shuttle box compartment farthest away from it at the time of CS presentation, the shock is presented (the Pavlovian component). After a number of CS-shock pairings, the honey bee makes the instrumental response of entering the compartment during the presentation of the CS, but before the onset of shock, thereby avoiding or postponing the shock (instrumental component). To perform a signalled avoidance experiment:

1. Select the parameters for the training variables. Table 3 provides a list.

2. Place the honey bee in the shuttle box for a 5 minute adaptation period.

3. Present the CS, if the honey bee does not respond by entering the compartment farthest away from it at any given time, present the shock. Do not present the shock if the honey bee responds during the presentation of the CS.

4. Measure on each CS presentation whether or not the bee entered the compartment farthest away from it (i.e., an avoidance response).

Fig. 6. Overview of a shuttle box. In this version, an aversive odour such as formic acid is used. The odour enters the device through the tubes shown in the bottom of the drawing. The bee walks on a screen floor and its movement is detected with infrared photo-detectors represented by the open circles. Lights located on the ceiling provide a cue signalling the onset of the aversive stimulus. The aversive stimulus can be terminated or avoided when the bee activates the photo-detector farthest away from it at any given time. To cross from one side of the chamber to another, the bee passes through a "tunnel" represented by the semi-circle.

1293PN revised Fig 6

Table 2.
Different categories of aversive conditioning in honey bees using a shuttle box.

category of aversive conditioning




fear conditioning

application of electric shock

shock remains on until bee leaves the current compartment

shock turns on when bee enters the other compartment

shock is omitted when bee enters compartment

response-independent pairings of light with shock

relationship between requisite response and shock






Abramson, 1986;

Agarwal et al., 2011

Agarwal et al., 2011

Abramson, 1986; Agarwal et al., 2011



Table 3. Variables affecting aversive conditioning.

training variable


- type of aversive stimuli

(electric shock, olfactory, temperature, vibration, noise, agrochemicals)

- type of shock (alternating or direct current)

- subspecies

affects all responses

- intensity of warnings and aversive stimuli

- timing of aversive stimuli

function is an inverted "U" shape

- duration of aversive stimulus

short durations are often more effective than longer durations

- schedule of aversive stimuli

continuous reinforcement schedules are more effective than intermittent schedules; a single presentation has different effects from a series of short bursts

- prior exposure to aversive stimuli

prior habituation or sensitization influence performance

- interval between CS and US stimuli

the shorter the interval, the more effective the association between a conditioned stimulus (CS) and an aversive unconditioned stimulus (US)

- delay between response and consequence

the shorter the delay the more effective the conditioning

- control of aversive stimuli by the bee or independent of the bee

the aversive stimulus can be terminated either by the bee or independently depending upon the paradigm

- magnitude of a negative reinforcer

frequency of escape responses is a function of reinforcement magnitude

- shock-shock (S-S) and response-shock (R-S) intervals in unsignalled avoidance

if the S-S and R-S intervals are short the response rate will be high, if these intervals are long, the response rate will be low

- type of extinction procedure

if the aversive event is omitted during extinction, performance will change faster than if the response and consequence, are unpaired