BEHAVIOR
Does Honey Bee Sting Alarm Pheromone Give Orientation
Information to Defensive Bees?
BROOK R. WAGER
AND
MICHAEL D. BREED
Department of Environmental, Population and Organismic Biology, The University of Colorado,
Boulder, CO 80309 Ð 0334
KEY WORDS Apis mellifera, honey bee, alarm pheromone, colony defense, orientation
AN IMPORTANT UNRESOLVED issue in understanding
honey bee defensive responses is the possible role of
the alarm pheromone in orienting bees to defensive
targets. Alerted honey bees could orient by chemotaxis to enemies marked by sting alarm pheromone
(Boch et al. 1962, Gary 1975, Winston 1987, p. 135).
Alternatively, alerted bees may target movement and
not use chemodirectional information (Free 1961,
Ghent and Gary 1962).
The question of the speciÞc role of alarm pheromone in target localization Þts into a larger picture of
understanding the functions of multicomponent pheromones. The honey bee sting alarm pheromone contains more than 20 compounds, although fewer than
half of them are active in releasing defensive behavior
in honey bee workers (Boch et al. 1962; Blum et al.
1978; Blum 1982, 1984; Pickett et al. 1982; Collins and
Blum 1983; reviewed by Free 1987). Sting alarm pheromone components have been tested for their roles in
alerting worker bees (Collins 1981; Collins and Blum
1982, 1983) and releasing stinging behavior (Free et al.
1983, 1987; Al-SaÕad et al. 1987). However, the role of
each component in the blend in the organization of
the defensive response (recruitment, ßight, orientation) is not well understood. Our elucidation of these
roles helps us to reÞne hypotheses regarding the evolution of this multicomponent blend.
The defensive response is usually initiated by guard
bees, which patrol the nest entrance and are the Þrst
line of colony defenders (Moore et al. 1987). Sting
deposition releases alarm pheromone, which causes a
defensive response; alarm pheromone alerts individuals, increasing their locomotion (Collins and Ku-
basek 1982) and recruiting bees from the interior of
the colony to the exterior (Boch et al. 1962, Collins and
Kubasek 1982, Collins and Blum 1983). Alarm pheromone may also be released by guards before stinging.
Bees are then attracted to the source of the disturbance by chemical or visual cues. Certain characteristics of the intruder, including movement, odor, and
color, may facilitate localization of the target by a
defending bee (Free 1987; Free et al. 1989).
In a previous attempt to partition the behavioral
roles of the alarm pheromone components, Free et al.
(1983, 1985) found that some compounds attract
whereas others repel bees. The Free et al. (1983)
Þndings demonstrate many of the difÞculties of working with highly volatile pheromones; concentration
may determine whether a compound is attractive or
repellent, and concentration is very difÞcult to control. Free et al. (1983) appropriately tested generalized behavioral responses to alarm pheromones but
did not adequately test the hypothesis that alarm pheromone gives orientational information to bees.
Of the alarm pheromone components, iso-pentyl
acetate (IPA, also called iso-amyl acetate) is the best
known. Collins et al. (1989) found positive correlations between levels of IPA and defensive behavior in
the Þeld (number of stings) and in laboratory tests,
Lensky et al. (1995) found that increasing levels of
stinging responses of isolated bees corresponds to the
concentration of IPA used for stimulation.
If IPA alone seems to account for much of the honey
bee alarm response, then why has such a complex
pheromonal mixture evolved? Multicomponent signaling systems, such as honey bee alarm pheromone,
0013-8746/00/1329Ð1332$02.00/0 䉷 2000 Entomological Society of America
Downloaded from https://academic.oup.com/aesa/article/93/6/1329/161481 by guest on 02 October 2023
Ann. Entomol. Soc. Am. 93(6): 1329Ð1332 (2000)
ABSTRACT We tested compounds found in honey bee, Apis mellifera L., sting alarm pheromone
for their roles in releasing behavioral responses, with a focus on the relative importance of chemotaxis
and motion of the target in the localization response. Some compounds in the blend have specialized
functions. Benzyl acetate released only ßight behavior, whereas three compounds (1-butanol,
1-octanol, and hexyl acetate) caused only the recruitment response. Other compounds (1-hexanol,
butyl acetate, iso-pentyl acetate, and 2-nonanol) acted in more than one behavioral context. Octyl
acetate was the most effective compound in allowing bees to locate targets, but did not recruit or
release ßight behavior. Stationary octyl acetate sources were located by ßying bees, indicating that
this pheromone component elicits a chemotactic response. However, localization of a target is due
primarily to the motion of the target; the alarm pheromone components release searching behavior
for a moving object and are relatively unimportant in target localization.
1330
ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA
Materials and Methods
Recruitment. The number of bees outside the hive,
on the entrance board and ßying near the entrance, is
an index of the magnitude of alarm response in honey
bees (Collins and Kubasek 1982). We photographically recorded alarm recruitment following Collins
and KubasekÕs (1982) methods. The following alarm
pheromone components were tested, as well as entire
stings: IPA, 1-hexanol, octyl acetate, butyl acetate,
2-methyl-l-butanol, 3-methyl-1-butanol, 1-butanol,
1-octanol, hexyl acetate, 2-nonanol, and benzyl acetate. A cotton-ball with no pheromone was placed on
the entrance to the hive (control). After 60 s, a photograph was taken with a D-300L Olympus Digital
Camera. Then 0.1 ml of the compound, a large enough
quantity to ensure olfactory exposure of the bee, was
placed on the cotton-ball, and after 60 s, another photograph was taken. Five colonies were tested, each
twice for each compound. Repeated tests on colonies
were separated by at least 2 d, to avoid carryover
effects among tests. Although the repeated tests were
not independent, with regard to possible genetic effects on responsiveness, they are independent tests of
the hypothesis that bees in this population respond to
each alarm pheromone. The number of bees present
in each of the photographs was recorded and the results
were analyzed with a paired t-test for equality of means.
Flight. Flight activity of isolated bees was measured
in an assay for rate of movement. A single bee was
placed in a glass chamber (30 by 45 by 75 cm) with a
line on center of the lid. A cotton-ball with 0.1 ml of
the compound was placed in the center of the container. The control was identical except a clean cotton-ball was placed in the center of the container. The
chamber was thoroughly ßushed with fresh air between replicates. In each replicate we recorded the
number of times a guard bee crossed the line, ßying,
in 60 s. Ten bees were used for each compound, as well
as the control; the results were analyzed with a paired
t-test for equality of means.
Localization. Two bioassays tested the orientation
of bees to each compound. First, single guard bees
Table 1. Number of bees responding to alarm pheromone
components at the hive entrance
Iso-pentyl acetate
1-hexanol
Octyl acetate
Butyl acetate
1-butanol
1-octanol
2-methyl-1-butanol
3-methyl-1-butanol
Hexyl acetate
2-nonanol
Benzyl acetate
Control
Control
mean ⫾ SD
Treatment
mean ⫾ SD
P
16.8 ⫾ 23.82
37.1 ⫾ 46.59
21.1 ⫾ 11.74
31.0 ⫾ 39.34
34.3 ⫾ 37.14
17.1 ⫾ 8.41
18.9 ⫾ 7.95
33.3 ⫾ 48.29
17.5 ⫾ 8.30
17.6 ⫾ 10.04
10.10 ⫾ 3.51
19.3 ⫾ 11.87
84.5 ⫾ 89.43
73.3 ⫾ 69.02
22.6 ⫾ 8.64
90.80 ⫾ 66.32
46.3 ⫾ 51.24
22.1 ⫾ 9.69
24.0 ⫾ 12.03
55.6 ⫾ 73.51
27.2 ⫾ 10.62
22.50 ⫾ 10.78
12.0 ⫾ 4.76
23.6 ⫾ 8.95
⬍0.05
⬍0.01
NS
⬍0.01
⬍0.05
⬍0.01
NS
NS
⬍0.01
⬍0.01
NS
NS
The mean number of bees before and after a pheromone treatment
on the entrance board (10 replicates, two at each of Þve colonies);
control and treatment means are compared using a paired t-test; NS,
not signiÞcant.
were placed in a T-shaped tube 22.5 cm across the top
with a 7.5 cm long stem made from 3-cm-diameter
clear plastic tubing. Each compound was presented by
exposing a small droplet at the tip of a 1.0-ml syringe.
Water was presented similarly as a control in the other
arm. The compound diffused down the long axis of the
tube. A cork was placed on the entrance to prevent the
bee from exiting without choosing either of the two
arms. Because of the possible confounding effect of
footprint pheromones (marking compounds putatively left by bees when they walk; Free 1987), the
tubes were thoroughly cleaned with solvents after
each test. We analyzed, using chi-square tests,
whether or not the bees exited toward the compound
more often than away from the compound. Thirty bees
were tested for each of the components.
In the second bioassay we investigated the interaction between motion of a target and orientation to
pheromone in the Þeld. A compound (0.1 ml) was
presented to the bees on a stationary cotton-ball suspended from a 34-cm rod near to (within 1 m) a colony
entrance. A clean cotton-ball, 65 cm from this target,
was set in motion on a 34-cm pendulum. Localization
was deÞned as a bee landing on the cotton-ball or
coming within 2.5 cm of the cotton-ball during ßight.
The number of times that bees localized to each target
was recorded for 5 min. Three trials were performed
for each compound. Five trials, using the same
method, tested the compound on the moving rather
than the stationary target. A sting (removed from a bee
and immediately presented on the apparatus) was
used as a positive control in this experiment.
Results
Recruitment. Seven of the 10 compounds induced
signiÞcantly more bees to exit the colony. These compounds were IPA, butyl acetate, 1-hexanol, 1-butanol,
1-octanol, hexyl acetate, and 2-nonanol (Table 1).
Flight. Three compounds, IPA, 2-nonanol, and benzyl acetate, caused signiÞcantly different levels of
ßight activity when compared with controls (Table 2).
Downloaded from https://academic.oup.com/aesa/article/93/6/1329/161481 by guest on 02 October 2023
are common among animals. The seemingly redundant information in complex signals helps animals to
segregate false messages, such as pheromones from
other species, from relevant signals. For social animals,
complex signals may help in coordinating group actions, in which different animals are responsible for
various aspects of the group response. The hypothesis
that the honey bee alarm pheromone has this type of
coordinating function underlies our experimental approach.
Here, we report results concerning three responses
to sting alarm pheromone: recruitment, locomotion,
and localization. Our primary focus was on the previously unresolved localization hypothesis; we tested
the ability of bees to locate each of 11 alarm pheromone components, as well as stings, in a t-maze. We
also assessed relative importance of motion and pheromone in attracting defensive bees.
Vol. 93, no. 6
November 2000
WAGER AND BREED: ALARM PHEROMONE ORIENTATION
1331
Table 2. Effect of alarm pheromone components on flight
activity of confined individual bees
Treatment
mean ⫾ SD
P
5.5 ⫾ 3.06
10.3 ⫾ 7.53
7.1 ⫾ 3.21
9.8 ⫾ 6.34
12.2 ⫾ 6.76
15.3 ⫾ 13.16
13.6 ⫾ 3.38
10.5 ⫾ 11.80
15.2 ⫾ 11.29
10.6 ⫾ 10.09
9.5 ⫾ 6.50
10.2 ⫾ 5.81
10.6 ⫾ 6.10
6.4 ⫾ 4.38
7.4 ⫾ 4.35
10.3 ⫾ 6.33
11.0 ⫾ 6.98
13.1 ⫾ 8.05
11.4 ⫾ 9.96
6.3 ⫾ 5.48
8.8 ⫾ 5.37
3.7 ⫾ 3.97
4.3 ⫾ 3.50
8.5 ⫾ 5.36
⬍0.05
NS
NS
NS
NS
NS
NS
NS
NS
⬍0.05
⬍0.01
NS
Means represent 10 guard bees; control and treatment means are
compared using a paired t-test; NS, not signiÞcant.
Of these, only IPA induced a higher level of ßight activity; 2-nonanol, and benzyl acetate signiÞcantly inhibited ßight. We observed no localization to the odor
source in the replicates in which ßight activity was inhibited; the bees did not land on or near the odor source.
Localization. In the t-tube tests, one compound,
2-nonanol signiÞcantly repelled bees (␹2 ⫽ 8.53, df ⫽
2, P ⫽ 0.0031). None of the compounds tested using
this method produced signiÞcant attraction, nor was
there a signiÞcant attraction to an isolated sting presented in the same fashion.
In the Þeld tests, only octyl acetate attracted bees
when placed on the stationary target (Fig. 1A). Some
of the compounds on the stationary target, such as
butyl acetate, actually induced more bees to approach
the moving target than the stationary target. Five of
the 10 compounds (iso-pentyl acetate, 1-hexanol, octyl acetate, n-pentyl acetate, butyl acetate), when
placed on the moving target, induced a signiÞcant
degree of localization, as did a sting (Fig. 1B).
Discussion
The major functions of honey bee alarm pheromone in colony defense are increasing ßight activity
(IPA), recruiting bees from the interior of the colony
(IPA, butyl acetate, 1-hexanol, 1-butanol, 1-octanol,
hexyl acetate, and 2-nonanol), and enhancing response to moving objects (IPA, 1-hexanol, octyl acetate, n-pentyl acetate, butyl acetate). Only 2-nonanol
gave orientational information in the t-tube apparatus, and only octyl acetate was attractive to bees
when not combined with motion. Single stings did not
provide localization information in either bioassay.
We conclude that the primary orientational role of
alarm pheromone is to alert bees and attract them to
moving objects (or animals) and that it is probably
inaccurate to assert a primary role for localization
through chemotaxis for alarm pheromone. Our data
clearly support the argument that motion is an important additional orientational stimulus (Free 1961, Collins and Kubasek 1982).
Our recruitment test results are largely in agreement with the most important previous studies on this
Fig. 1. Number of bees responding to chemical stimuli on
targets near colony entrances. (A) Chemical was placed on
the moving target. Solid bars are means (n ⫽ 5) of the number
of bees approaching the moving target, open bars are means
of the number of bees approaching the stationary target (the
two targets were simultaneously present). (B) Chemical was
placed on the stationary target. Solid bars are means (n ⫽ 3)
of the number of bees approaching the stationary target,
open bars are means of the number of bees approaching the
moving target (the two targets were simultaneously present).
The 13 compounds and treatments are as follows: (1) isopentyl acetate, (2) 1-hexanol, (3) octyl acetate, (4) butyl
acetate, (5) 3-methyl-1-butanol, (6) 1-butanol, (7) 1-octanol,
(8) 2-methyl-1-butanol, (9) hexyl acetate, (10) 2-nonanol,
(11) benzyl acetate, (12) control, (13) sting apparatus.
topic (Collins and Kubasek 1982, Collins and Blum
1983, Moritz et al. 1985). Our study, as well as Collins
and Blum (1983), showed a strong alarm response,
marked by an increase in numbers of bees responding,
caused by 1-hexanol, compared with intermediate responses from IPA and 1-butanol.
Free (1987, p. 138) summarized his conclusions
concerning the functions of the alarm pheromone
Table 3. Summary of the results from the bioassays for alarm
pheromone component effects on flight, recruitment, and
localization
Flight
activity
Iso-pentylacetate
1-hexanol
Octyl acetate
Butyl acetate
1-butanol
1-octanol
2-methyl-1butanol
3-methyl-1butanol
Hexyl acetate
2-nonanol
Benzyl acetate
Localize
Localize
Recruitment stationary
moving
compound compound
Increase
No effect
No effect
No effect
No effect
No effect
No effect
Yes
Yes
No effect
Yes
Yes
Yes
No effect
No effect
No effect
Yes
No effect
No effect
No effect
No effect
Yes
Yes
Yes
Yes
No effect
No effect
No effect
No effect
No effect
No effect
No effect
No effect
Decrease
Decrease
Yes
Yes
No effect
No effect
No effect
No effect
No effect
No effect
No effect
Downloaded from https://academic.oup.com/aesa/article/93/6/1329/161481 by guest on 02 October 2023
Iso-pentyl acetate
1-hexanol
Octyl acetate
Butyl acetate
1-butanol
1-octanol
2-methyl-1-butanol
3-methyl-1-butanol
Hexyl acetate
2-nonanol
Benzyl acetate
Control
Control
mean ⫾ SD
1332
ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA
Acknowledgments
This study was supported by a grant from the National
Science Foundation (USA), IBN 9408180 and a grant to
B.R.W. from the Undergraduate Research Opportunities
Program at the University of Colorado at Boulder.
References Cited
Al-Sa’ad, B. N., J. B. Free, and P. E. Howse. 1987. Adaptation
of worker honey bees (Apis mellifera) to their alarm
pheromones. Physiol. Entomol. 10: 1-4.
Blum, M. S., H. M. Fales, K. W. Tucker, and A. M. Collins.
1978. Chemistry of the sting apparatus of the worker
honeybee. J. Apicult. Res. 17: 218-221.
Boch, R., D. A. Shearer, and B. C. Stone. 1962. IdentiÞcation
of iso-amyl acetate as an active compound in the sting
pheromone of the honeybee. Nature (Lond.) 195: 10181020.
Collins, A. M. 1981. Effects of temperature and humidity on
honeybee response to alarm pheromones. J. Apicult. Res.
20: 13-18.
Collins, A. M., and M. S. Blum. 1982. Bioassay of compound
derived from the honeybee sting. J. Chem. Ecol. 8: 463470.
Collins, A. M., and M. S. Blum. 1983. Alarm responses
caused by newly identiÞed compounds derived from the
honey bee sting. J. Chem. Ecol. 9: 57-66.
Collins, A. M., and K. J. Kubasek. 1982. Field test of honey
bee (Hymenoptera: Apidae) colony defense behavior.
Ann. Entomol. Soc. Am. 75: 383-387.
Collins, A. M., T. E. Rinderer, and H. V. Daly. 1989. Alarm
pheromone production by two honey bee (Apis mellifera) types. J. Chem. Ecol. 15: 1747-1756.
Free, J. B. 1961. The stimuli releasing the stinging response
of honey bees. Anim. Behav. 9: 193-196.
Free, J. B. 1987. Pheromones of social bees. Cornell University Press, Ithaca, NY.
Free, J. B., A. W. Ferguson, J. R. Simpkins, and B. N. Al-Sa’ad.
1983. Effect of honeybee nasanov and alarm pheromone
components on behaviour at the nest entrance. J. Apicult.
Res. 22: 214-223.
Free, J. B., J. A. Pickett, A. W. Ferguson, J. R. Simpkins, and
M. C. Smith. 1985. Repelling foraging honeybees with
alarm pheromones. J. Agric. Sci. Cambr. 105: 255-260.
Free, J. B., A. W. Ferguson, and J. R. Simpkins. 1989. Honeybee responses to chemical-components from the
worker sting apparatus and mandibular glands in Þeldtests. J. Apicult. Res. 28: 7-21.
Gary, N. E. 1975. Activities and behavior of honey bees. In
The hive and the honey bee. Dadant & Sons, Hamilton, IL.
Ghent, R. L., and N. E. Gary. 1962. A chemical alarm releaser in honeybee stings. Psyche 69: 1-6.
Lensky, Y., P. Cassier, and D. Tel-Zur. 1995. The setaceous
membrane of honey bee (Apis mellifera) workers sting
apparatus: structure and alarm pheromone distribution.
J. Insect Physiol. 41: 589-595.
Moore, A. J., M. D. Breed, and M. J. Moor. 1987. The guard
honey bee: ontogeny and behavioral variability of workers
performing a specialized task. Anim. Behav. 35: 1159-1167.
Moritz, R.F.A., E. E. Southwick, and M. Breh. 1985. A metabolic test for the quantitative analysis of alarm behavior
on honeybees (Apis mellifera L.). J. Exp. Zool. 235: 1-5.
Pickett, J. A., I. H. Williams, and A. P. Martin. 1982. (Z)11-eicosen-1-ol, an important new pheromonal component from the sting of the honey bee, Apis mellifera, L. J.
Chem. Ecol. 8: 163-175.
Winston, M. L. 1987. The biology of the honey bee. Harvard
University Press, Cambridge, MA.
Received for publication 27 January 2000; accepted 22 August 2000.
Downloaded from https://academic.oup.com/aesa/article/93/6/1329/161481 by guest on 02 October 2023
components from the mandibular glands and the sting.
Although most of the functions he considers are related to foraging and scenting, two parameters correspond to the tests for ßight and recruitment performed
in this study. The results of our study correspond with
his Þndings. However, butyl acetate, hexyl acetate,
and octyl acetate, shown by Free (1987) to be significant activators of caged bees, did not induce significant increase of ßight by an isolated bee in our study.
Perhaps their role is in other aspects of the alarm response, rather than induction of higher levels of ßight
activity.
Despite the presence of some response speciÞcity in
our data, most of the compounds that we tested were
active in more than one behavioral category, and we
conclude that there is no well-deÞned division of
functions among the compounds in this multicomponent pheromone (Table 3). One component that we
did not test, (Z)-11-eicosen-1-ol (Pickett et al. 1982)
is much less volatile than those that we tested. Although previous tests of the role of eicosenol do not
suggest an arrestant role for that compound, it would
be interesting in future work to pursue the possibility that a subset of compounds, perhaps including
2-nonanol, benzyl acetate, and eicosenol have an arrestant (rather than an orientational) function.
Do our results help us to understand the roles of
multicomponent signals in segregating irrelevant from
relevant signals (through redundancy) and in coordinating social responses? Clearly, there is considerable redundancy in the responses we observed, with
a large number of compounds being active in recruitment and a similar number being active in stimulating
visual localization. However, the individual compounds elicit strong responses even when not presented in the context of the blend. We do not reject
the idea that redundancy in multicomponent signals
may reduce the risk of a false response, but in the case
of honey bee alarm pheromone other explanations of
signal redundancy may be needed. The differentiation
among chemicals in the type of response elicited
points to social coordination as a possible important
function of this multicomponent pheromone.
Finally, although individual sting pheromone components may stimulate speciÞc behaviors, this does not
necessarily eliminate the apparently inactive compounds as important functional components in the
pheromone complex. They may have synergistic functions or speciÞc combinations of compounds may induce speciÞc behaviors. The number of compounds
and their many possible effects make this an exceedingly complex system.
Vol. 93, no. 6