Training Phase
60 adult female crickets were selected at random from our
aquarium and were equally distributed into four large containers. Each
container represented a distinct condition. A summary of the
conditions is displayed in Figure 1, and the experimental setup is
depicted in Figure 2a. In each container, the sole food sources were
two food dishes, across from each other. Both dishes were composed
of commercial cricket food. One dish was 1% quinine, a substance
aversive to the cricket (Matsumoto and Mizunami 2000), while the
other did not contain quinine; these served as the aversive and
appetitive unconditioned stimuli (US), respectively. Each dish also
contained either cinnamon or cumin, in order to give each food dish a
distinct odour. 10 grams of spice was used for every 100 grams of
food. One of a pair of odours (cinnamon/cumin), food dish colours
(brown/green), and spatial locations (left side/right side) were used as
conditioned stimuli (CS) for each of the two dishes.
The only combination of CSs that remained constant throughout
every condition was that brown food dishes always smelled like
cinnamon, and green food dishes were always associated with cumin.
We counterbalanced in 3 ways. Half of the crickets were trained with
the appetitive US on the left side and the aversive US on the right side,
and vice-versa. Cinnamon served as a CS for the appetitive US for half
of the crickets, but was a CS for the aversive US for the other half, and
the same was true for cumin. Similarly, the cinnamon-scented food
was on the left side for half the crickets, and on the right side for the
other half; again, the same was true for the cumin-scented food.
For 7 days, the crickets were allowed water and fed this diet ad
libitum. Then we randomly selected 8 crickets from each container
and moved them into individual containers. The crickets were starved
for three days in order to enhance their motivation to search for food in
the testing phase.
Testing Phase
Each trial began when we placed two food dishes into each
container across from each other at one end. Other than the fact that
neither food contained quinine, the cue combinations were consistent
with those from training. Figure 2b illustrates the experimental setup.
Crickets were allowed 15 minutes to eat from one of the two dishes.
Two blind observers recorded which of the two dishes each cricket
initially chose to eat, and we considered this dish to be the one that
they preferred. If the cricket did not eat within 15 minutes, we
considered the cricket to have made no choice, and did not include
these data points in our statistical analyses.
Figure 2. A simplified version of the experimental setup. Note that the specific combination
of stimuli presented is unique to one of the 4 conditions. a. Crickets were trained to
associate one of a pair of odours, colours, and spatial locations with food containing quinine,
and the other pair member with non-aversive food. Note that the cricket in b. was trained to
associate cinnamon, brown, and the left side with quinine this was done to better illustrate
the relationship of the stimuli presented in both phases of the experiment. There were also
15 crickets and pieces of egg carton in each container, but they were omitted for clarity. b.
The stimuli presented to each cricket differed from that from training solely in that neither
dish contained quinine. Egg cartons were also present in the cage, but were omitted for
clarity.
Data Analysis
A binomial test was used to determine if the proportion of crickets
that preferred the dish that had appetitive cues was significant. We
used a Fishers Exact Test to determine whether there was a significant
difference in performance depending on the combination of CSs with
which a cricket was trained. Two more binomial tests were conducted
Figure 3: Crickets were trained to associate an odour/side pairing with an aversive dish, and
another pairing with an appetitive dish. Possible combinations of CSs associated with the
aversive US were cinnamon and the left side (Cinnamon/L -), cinnamon and the right side
(Cinnamon/R -), cumin and the left side (Cumin/L -), and cumin and the right side (Cumin/R
-). Shown are the proportions of crickets in each condition (and overall) who preferred the
dish that had the appetitive cues (proportion learned).