Abstract The invertebrate neuromodulator octopamine is known to be involved in bees&apos; associative learning, enhancing the responsiveness of a bee to a conditioned stimulus. In this study, we tested the effect of octopamine on the choice behavior of free-flying bumblebees using a two-phase experiment in an array of artificial flowers. During the first phase of the experiment, the bee was allowed to collect octopamine-laden sugar water from two types of equally rewarding flowers (yellow versus blue). In the second phase, one type of flower was set to be unrewarding. The behavior of the bee (proportion of visits to the unrewarding flowers) over the two phases was fitted to a sigmoid regression model. Our results show that octopamine had no significant effect on the bees&apos; equilibrium choice or on the overall rate of the behavioral change in response to the change in reward. Rather, octopamine significantly affected the time interval between the change in reward status and the initiation of behavioral change in the bee

Dan R Papaj

Jonathan Cnaani

Justin O Schmidt

CiteSeerX

Naturwissenschaften (2003) 90:185–188
DOI 10.1007/s00114-003-0412-9
S H O R T C O M M U N I C A T I O N
Jonathan Cnaani · Justin O. Schmidt · Dan R. Papaj
The effect of octopamine on behavioral responses
of free-foraging bumblebees to a change in food source profitability
Received: 2 September 2002 / Accepted: 21 February 2003 / Published online: 25 March 2003
 Springer-Verlag 2003
Abstract The invertebrate neuromodulator octopamine is
known to be involved in bees’ associative learning,
enhancing the responsiveness of a bee to a conditioned
stimulus. In this study, we tested the effect of octopamine
on the choice behavior of free-flying bumblebees using a
two-phase experiment in an array of artificial flowers.
During the first phase of the experiment, the bee was
allowed to collect octopamine-laden sugar water from two
types of equally rewarding flowers (yellow versus blue).
In the second phase, one type of flower was set to be
unrewarding. The behavior of the bee (proportion of visits
to the unrewarding flowers) over the two phases was
fitted to a sigmoid regression model. Our results show
that octopamine had no significant effect on the bees’
equilibrium choice or on the overall rate of the behavioral
change in response to the change in reward. Rather,
octopamine significantly affected the time interval be-
tween the change in reward status and the initiation of
behavioral change in the bee.
Introduction
Neuromodulators such as biogenic amines and peptides
are substances that elicit and modify behavior patterns.
They orchestrate behavior by acting simultaneously at
many synaptic levels in both the central and peripheral
nervous system, and also by a broader regulation of
metabolism (Bicker and Menzel 1989; Roeder 1999). In
social insects, one biogenic amine, octopamine, has been
implicated in a variety of behaviors. In honeybees, for
instance, octopamine is involved in the regulation of
division of labor (Schulz and Robinson 1999); oral
administration of octopamine to honeybee workers results
in increased foraging activity (Schulz and Robinson
2001). Octopamine is likewise thought to be involved in
the regulation of trophallaxis in ants and the establish-
ment of social status in bumblebees (Bloch et al. 2000;
Boulay et al. 2000).
One intensively studied behavior in bees for which the
role of octopamine has not been evaluated to date is floral
choice. One reason to anticipate a role for octopamine in
the context of floral choice is that, in generalist bees,
floral preferences are formed and modified through
learning. In such bees, there is ample evidence that
octopamine is involved in cue learning with nectar
rewards. Mercer and Menzel (1982), for example, showed
that responsiveness to the conditioned stimulus (CS), in
this case orange scent, in a proboscis extension reflex
(PER) conditioning paradigm was enhanced by octopam-
ine, closely resembling the effect of sensitization. Later,
Hammer (1993) identified a neuron in the subesophageal
ganglion of the honeybee, termed VUMmx1, which
mediated the reinforcing function of sugar in odor-
conditioning of the proboscis extension response. This
neuron belongs to a group of octopamine-immunoreactive
neurons (Kreissl et al. 1994), and it was consequently
hypothesized that octopamine is released by VUMmx1
under sugar stimulation and mediates the effect of CS
reinforcement in PER odor conditioning. In support of
this hypothesis, Hammer and Menzel (1998) showed that
the reinforcing effects of sugar stimulation could be
substituted by odor-coupled injection of octopamine into
either of two brain regions, the antennal lobe or the
calyces of the mushroom bodies.
Despite the detailed description of the role of octo-
pamine in associative learning in bees under highly
J. Cnaani ()) · J. O. Schmidt
Carl Hayden Bee Research Center,
USDA-ARS, 2000 E. Allen Rd., Tucson, AZ 85719, USA
e-mail: jcnaani@zoo.utoronto.ca
Tel.: +1-416-9783507
Fax: +1-416-9788532
D. R. Papaj
Department of Ecology and Evolutionary Biology,
The University of Arizona,
Tucson, AZ 85721, USA
Present address:
J. Cnaani, Department of Zoology,
University of Toronto,
25 Harbord St., Toronto, ON M5S 3G5, Canada
controlled conditions, little is known about how octo-
pamine affects the more complex choice behavior exhib-
ited by free-foraging animals. To address this question,
we trained individual bumblebee workers to collect
octopamine-laden sugar water from six artificial flowers
of two colors, blue and yellow. Our experiment was not
designed to identify a specific mechanism by which
octopamine affects choice behavior, or specific sites in
the brain involved in an effect on behavior. Rather, we
attempted to define in detail at the effect of octopamine
on the behavior of the free-foraging bee. We view this
approach as complementary to studies carried out in
restrained bees (see Mercer and Menzel 1982; Hammer
1993; Hammer and Menzel 1998), which concentrate on
specific sites of action and neurological pathways where
octopamine affects associative or non-associative learn-
ing.
Methods
The experiment took place in a small flight chamber
(1.93.01.8 m) illuminated only by fluorescent light, in Octo-
ber–November 2001. A commercial bumblebee hive (purchased
from Koppert Biological Systems) was placed outside the chamber,
and foragers were allowed to enter the chamber through a short
tube. During the experiment, only one forager was allowed into the
room, but during all other times, bees had free access to the flight
chamber and were allowed to feed on sugar water (30%) from two
feeders placed on the experimental table. Temperature in the room
ranged between 25 and 30C. Artificial flowers (three blue and
three yellow) were arranged as a circle of alternating colors around
the edge of a round table (diameter 1.2 m). Each “flower” consisted
of a small container that holds about 10 ml of sugar water. A cork
with a magnetic collar floats on the sugar solution and prevents
access by the bee. The bee instead has access to a small cup (4 l)
mounted on top of the cork. This cup is filled with sugar water by
activating an electromagnet, which pulls the cup down into the
container. The cup is filled with sugar water and, upon deactivating
the electromagnet, returned to its original position, a process that
takes 3 s to complete. Refilling of each of the six flowers is
controlled at a user-specified time interval (termed the refilling
interval). A second simultaneously running program detects the
bee’s probing into flowers and records the exact time of probing
and the specific flower visited.
Each individual bee (a total of 39, all from the same colony)
was tested in a two-phase experiment. In the first phase, the two
types of flowers were equally rewarding. The refilling interval was
set to 60 s for all six feeders and each bee was allowed to complete
180 visits (this particular time interval between refilling of the
flowers allows the foraging bee to find a rewarding flower in about
70% of her visits). During this phase, the bee learned how to collect
sugar water from the feeders. In the second phase, the three yellow
feeders were turned off (gave no reward) and the refilling interval
for the three blue feeders was set to 30 s. At these values, the
overall availability of sugar water in the system was the same as in
the first phase, but the two color types differed in reward. The bee
was allowed to perform an additional 420 visits, and the change in
the proportion of visits to the yellow feeders was recorded.
Completing the 600 visits (both phase 1 and 2) required a bee to
perform an average of 19.5 bouts (about 30 visits each) and took
about 2 h to complete.
Using this paradigm, the pattern in floral choice was determined
for bees fed variable levels of octopamine. Octopamine (Sigma)
was dissolved in sugar water (30% sucrose) at concentrations of 0,
2, 5, and 8 mg/ml [a concentration range used by Schulz and
Robinson’s (2001) study on honeybees]. Feeders were then filled
with octopamine-containing sugar water of the desired concentra-
tion. Any given test bee was fed at one, and only one, level of
octopamine in all feeders. In addition to the four treatment groups
of bees tested over two phases, a control group of bees was allowed
to complete a series of 600 visits at 0 mg/ml of octopamine, with
the refilling intervals held constant at 60 s for both feeder types.
To analyze the behavioral response to the change in feeder
profitability, visits of individual bees were arbitrarily binned into
sets of 30 successive visits. For each set, the proportion of visits to
the yellow feeder was calculated. The relationship of the proportion
of visits to the yellow feeder (Y) to the number of visits made (X)
was analyzed using a sigmoid model:
Y ¼ Y0 þ
a
1þ e
XX0
b
 
where a is the amplitude, the distance between two asymptotes; b is
the rate of transition between the asymptotes; X0 is the value of the
inflection point, equal to the number of visits at which 50% of the
transitions between the two asymptotes have occurred; and Y0 is the
value of the second asymptote. This model was used because it
described very well the steady-state value of choice in each phase
as well as the transition from one steady-state value to the other.
Preference under steady-state conditions is described by Y0, the
steady-state value of choice in the second phase, as well as Y0a
(the difference between Y0 and a), which is equal to the steady-state
value of choice in the first phase. An effect of octopamine on these
parameters thus reflects an effect on floral choice. The transition
between the two states is described by parameters b, the rate of
change from one state to the other, and X0, the inflection point of
the transition. An effect of octopamine on the b parameter reflects
an effect on learning while an effect of octopamine on X0 reflects an
effect on the bee’s arousal.
Results
During the first phase, when the refilling interval was
identical for blue and yellow feeders, the mean proportion
of visits to the yellow feeders was 0.51€0.02 (n=34).
Although this value is significantly different from the
expected proportion of visits (one sample t-test, P=0.007),
the deviation is so small that, practically speaking, the
bees visited the two types of flowers at almost equal
frequencies. Following the change in the refilling interval,
bees showed a gradual shift in the proportion of visits to
the now-unrewarding yellow feeders and, by the end of
the second phase, almost no visits were made to these
feeders (Fig. 1).
Using sigmoid regression analysis (SigmaPlot 4.0), the
four model parameters were estimated for each bee. The
effect of treatment (octopamine concentration in the sugar
water) on these estimates was tested using linear regres-
sion. Overall, only the effect of octopamine treatment on
inflection point was significant (Y0, the second asymptote,
F3,25=0.381, P=0.54; a, the amplitude of change in states,
F3,25=0.09, P=0.75; b, the rate of transition between the
two asymptotes, F3,25=0.014, P=0.91; X0, the inflection
point, F3,25=8.23, P=0.008). Thus, our results show that
octopamine does not affect the steady-state values of
preference (for example, by altering the perception of
differences in reward), but rather affects the manner in
which preference changes following a change in reward
schedule. In particular, octopamine affected the parameter
X0, the inflection point of the sigmoid relationship. Given
186
that octopamine did not affect either the asymptotic
values of preference or the rate of transition from one
asymptotic value to the other, the effect on inflection
point amounts to an effect on the initiation of transition
from the steady-state preference in the first phase. In
short, bees fed at higher levels of octopamine appear to
respond earlier to the change in reward schedule.
Discussion
Given these results, at least two questions arise. First,
does variation in the level of octopamine at the feeders
result in differences in octopamine levels in the brain?
Foragers were first exposed to octopamine when the
experiment began and testing began about 30 min after
the end of the first phase. The octopamine-laden sugar
water was carried by the forager to the colony in their
crop where no sugar is absorbed (Crailsheim 1988).
Nevertheless, according to Visscher et al. (1996), the
sugar reserves in the honeybee hemolymph (about 2 mg)
allow a worker to fly for about 12 min, after which it
releases some nectar from the crop into the midgut, where
the sugar is absorbed. If this value is similar for
bumblebees, then by the end of the first phase of the
experiment the forager is probably releasing some of the
octopamine sugar water from the crop into the midgut.
Schulz and Robinson (2001) used a similar technique to
administer octopamine to bees and to measure its effect
on division of labor in free-foraging honeybees. Using
HPLC analysis, they showed that bees fed sugar water
with octopamine had significantly higher levels of this
biogenic amine in the brain; however, it was not clear
whether the orally administered octopamine reached the
brain. Ingested octopamine may conceivably pass through
the gut and the blood–brain barrier; alternatively, it may
generate endocrine responses which stimulate octopamine
production in the brain. The bees in their experiments
were fed octopamine chronically for a few days; in our
experiment, bees were allowed to feed on octopamine-
laden sugar water for only about 30 min before tests
began (the approximate duration of phase 1). Given the
difference in time frame, and lacking information about
how long orally administered octopamine takes to reach
the brain, it would be desirable to assess octopamine titers
in the brain under our procedure.
A second question that needs to be addressed further is
whether or not the effect of octopamine observed here
reflects an effect on learning. As discussed above,
octopamine has been shown to have a dual effect on
learning in honeybees. It has a direct effect on associative
learning wherein it can substitute for the unconditioned
stimulus (Hammer and Menzel 1998), and an indirect
effect wherein it increases sensitization (Mercer and
Menzel 1982). Our results show no effect of octopamine
on acquisition (b and Y0 parameters in the sigmoid
equation); rather, octopamine affected the choice process
indirectly by shortening the time lag between the change
in profitability (when information about relative rewards
start to accumulate) and the initiation of behavioral
change. This may explain why the only affected param-
eter is the inflection point (X0). These findings are fully
consistent with the effect of brain-injected octopamine
found by Mercer and Menzel (1982) using the PER
paradigm with restrained honeybees.
Acknowledgements This research has been supported by a
research grant funded by the Binational Agriculture Research and
Development (BARD) grant No. IS-3024-98 (to J.O.S.) and by
Fig. 1 Change in the proportion
of visits to the three yellow
feeders. During the first 180
visits, all six feeders were re-
filled with sugar water every
60 s. After this period, the three
yellow feeders were shut-off
and did not refill again. The
three blue feeders were refilled
every 30 s. The figure presents
the change in the proportion of
visits to the yellow feeders for
different concentrations of oc-
topamine in the sugar water
187
BARD postdoctoral fellowship No. FI-286-99 (to J.C.). The
experiments in this study comply with the current laws of the
country in which the experiments were performed.
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The effect of octopamine on behavioral responses of free-foraging bumblebees to a change in food source profitability

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The effect of octopamine on behavioral responses of free-foraging bumblebees to a change in food source profitability

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