Impulsivity Experiment
Associative Conditioning PER
Each conditioning trial consisted of presenting a bee with a 3 second pulse of the odor as
conditioned stimulus (CS) followed by a given volume of 50% sucrose reward as an
unconditioned stimulus (US), with a 2 second overlap between the CS and the US. The delivery
system consisted of an air pump connected to a glass syringe, placed facing the bee 1 cm away,
and containing a filter paper soaked with 5 µl of odor, through which the air flow was regulated
by computer controlled valves. The odors, 1-Octanol, Eugenol, Hexanol, and Octanone, were
paired with the large and the small reward, the specific pairings being different across
experimental replicates to control for any odor biases. The two odor-reward pairings were
presented to each subject in a pseudorandom sequence of ABABBABAABABABAB for a total
of 16 consecutive conditioning trials, with an inter-trial interval of 11 min. If a bee extended its
proboscis to the CS before the US is presented, it was given a score of 1 for exhibiting the
conditioned response, otherwise it was given a score of 0. Any bee which did not repsond at least
once to each of the small and large reward odor in the conditioning training was excluded from
further analysis.
Two-alternative forced choice PER
Each of the two odors (CS) was presented twice, in two alternating 0.8 s non-overlapping pulses
with 0.2 s of odorless air in between successive odor pulses, in the sequence ABAB. The side
from which each odor was pulsed was changed across replicates to control for any side bias. All
choice tests were conducted blind, with the observer not being aware of the specific nature of the
odor-reward pairing.
1
Biogenic Amines Quantification
Any partial brain was discarded and biogenic amines were quantified on a per brain basis.
Sample preparation consisted of removing the hypopharyngeal gland and occipital lobes from the
brain during dissection and adding 50 µl of chilled 0.2 M perchloric acid to each tube containing
three brains. The perchloric acid contained 100 pg/µl of N-Methylserotonin oxalate and 50 pg/µl
of synephrine, which served as internal standards. N-Methylserotonin oxalate was used to
monitor the recovery of dopamine and serotonin, and synephrine was used to monitor
octopamine and tyramine. Each sample was macerated with a sterile pestle and then sonicated
for 5 min in ice water, and these samples were then re-chilled on ice for 20 minutes. The contents
were centrifuged at 15,000 g set at 4° C for 10 min, the supernatant was transferred to a 0.22-µm
nylon membrane filter microcentrifuge spin column and centrifuged again at the same speed for
6 min. The filtered sample was transferred to a dark amber microvial for HPLC analysis. All
standards were purchased from Sigma-Aldrich.
HPLC analysis was performed using a Thermo Scientific Dionex Ultimate 3000 HPLC
system consisting of a SR-300 solvent rack, a refrigerated ISO-3100BM pump, a WPS3000TBSL analytical autosampler maintained at 4° C, an 80 mm x 4.6 mm high-efficiency C18
reverse-phase catecholamine HR-80 column (3 μm particle packing) maintained at 32° C, and a
4-channel ECD-3000RS electrochemical detector. Only 2 of the channels on the electrochemical
detector were used, one channel was set to 650 mV for octopamine and tyramine, while the other
was set to 350 mV for dopamine and serotonin. The mobile phase was composed of 10%
acetonitrile, 75 mM sodium dihydrogen phosphate monohydrate, 1.7 mM 1-octanesulfonic acid
2
sodium salt, 25 µM EDTA tetrasodium tetrahydrate, and the pH was adjusted to 3 using
concentrated phosphoric acid. The flow rate of the mobile phase was 0.4 ml/min. External
standards were run at the beginning, middle, and end, and all the samples were run at the same
time, in a randomized order.
Statistical Analyses
A repeated measures binomial logit generalized linear mixed model (GLMM) was used
to evaluate the acquisition of the two odor-reward pairings, where trial number and reward size
were fixed factors, the experimental replicate and odor were random factors, and conditioned
responses scored as a 1 was the dependent variable. A repeated measures binomial logit GLMM
was used to analyze the preference for the large reward in the choice tests, with starvation time
and delay treatment as fixed factors, experimental replicate and odor as random factors, and the
head position towards the odor associated with the large reward scored as a 1 as the dependent
variable. A multivariate general linear model (GLM) was used to compare the amount of each
biogenic amine, with starvation time as the independent variable.
3
Figure S1. Acquisition curve for the two CS-US pairings, a 1 µl large reward linked with a 5 s
delay, and a 0.2 µl small reward linked with a 1 s delay. There is a significant effect of both trial
number (Repeated measures GLMM: F1,2,450 = 38.82, P < 0.0001) and reward value (F1,2,450 =
68.10, P < 0.0001) on the proportion of conditioned responses, with data consisting of 167 bees.
4