Journal of Apicultural Research

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The Effect of Citral and Geraniol Conditioning on

the Searching Activity of Honeybee Recruits

Gordon D. Waller

To cite this article: Gordon D. Waller (1973) The Effect of Citral and Geraniol Conditioning on
the Searching Activity of Honeybee Recruits, Journal of Apicultural Research, 12:1, 55-59, DOI:
10.1080/00218839.1973.11099730

To link to this article: https://doi.org/10.1080/00218839.1973.11099730

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 55

Journal of Apicultural Research 12(1) : 53-57 (1973)

THE EFFECT OF CITRAL AND GERANIOL

CONDITIONING ON THE SEARCHING ACTIVITY OF
HONEYBEE RECRUITS*

GORDON D. WALLER

Bee Research Laboratory, Agricultural Research Service, U.S. Department of

Agriculture, Tucson, Arizona 85719, U.S.A.

Revised manuscript received for publication 5th September /972

Summary
Ten forager honeybees from Caucasian and from Cordovan mutant Italian colonies in a wire-screen
flight cage were marked, and conditioned to collect sucrose solution placed above petri dishes scented
with citra! or geraniol. Recruits from colonies conditioned to citra! alone or to geraniol alone were
attracted to the training scent in greater numbers when it was presented alone than when in com-
bination with the other scent; very few were attracted to the other scent alone or to the unscented
dishes. A large number of recruits from a wild-type Italian colony conditioned to citra! + geraniol
were attracted to dishes with the combination of scents and fewer to dishes with a single scent.
Recruits from the same colony were not attracted to any of the scented dishes while its foragers were
conditioned to gather sucrose from an unscented training station.

Introduction
The importance of olfaction in recruitment of forager honeybees (Apis mellifera) is
well documented (Frisch, 1967; Johnson & Wenner, 1970), but a better understanding
of the way bees respond to the components of odours might help us in our attempts to
manipulate foraging behaviour. Koltermann (1969) showed that association with
successful foragers, i.e., social communication, was sufficient to direct 99% of recruits
to a dish with the training scent. The chemical and biological properties of the two
major components of the Nasonov pheromone, citra! and geraniol, have been studied
by several workers (literature reviewed by Waller, 1970). Waller found that more
honeybees visited alfalfa plots scented with citra! plus geraniol than visited plots
scented with only one of the two scents. This may have been (1) because citra! and
geraniol in combination are more attractive to individual foragers than either alone,
or (2) because some bees recruited by foragers from the single-scented plots went to
double-scented plots despite the presence of the additional unfamiliar scent. Rib bands
(1955) found that bees were less responsive to the addition of an unfamiliar scent
than to the absence of one of two scents to which they had been conditioned. Thus,
recruits arriving at the double-scented plots might have been recruited by foragers
from plots scented with either one of the scents or with the combination. The
experiment reported here was designed to test the second hypothesis.

Materials and Methods

The work was carried out at the Bee Research Laboratory, Tucson, Arizona, in
February 1970.
The test cage was 22x 19·5x2·75 m and made of wire screen that excluded all bees

* In co-operation with the Arizona Agricultural Experimental Station, University of Arizona, Tucson.
56

of unknown sources from the test area. One colony each of Caucasian, wild-type
Italian, and Cordovan mutant Italian honeybees, hereafter referred to as Caucasian,
Italian and Cordovan, respectively, was used. The Caucasian and Cordovan
colonies were placed in the NW and NE corners of the cage, respectively; the
Italian colony was placed outside the cage with entrances provided so that these bees
could be allowed to fly in the cage or outside (Fig. 1). The use of the Caucasian
and Cordovan bees, which represent the extremes in genetic markers, allowed the
development of the necessary techniques for conditioning and marking foragers*
and testing the response of recruits.

0 0 0 0

ltTSE)

E]cors CaTSEJ

FIG. I. Experimental layout within the flight cage, February 1970.

Ca : Caucasian colony; CaTS : Caucasian training station, scented with geraniol 7-12 Feb. and with
citra! 13-19 Feb.
Co : Cordovan colony; CoTS : Cordovan training station, scented with citra! 7-12 Feb. and with
geraniol 13-19 Feb.
It :Italian colony (entrance into cage or outside); ItTS :Italian training station, no scent provided
13-17 Feb., scented with citra!+ geraniol18-19 Feb.
LS : Latin square of scented dishes.

Ten foragers from each of two colonies (Cordovan and Caucasian) were conditioned
to feed at separate training stations, which consisted of tables 0·5 m square and 0·5 m
high each supporting a petri dish 5·5 X 1 em (Fig. 1). The petri dish contained a
filter paper disc soaked with 6-10 drops of paraffin oil which was used to hold and
dissipate the two training scents, citral or geraniol. When used alone, 20 111 of each
scent was added to the paraffin oil. Double-scented dishes contained 20 111 of each
scent on 9th and lOth February, but only 10 j.d on 17th and 19th February. A wire
screen placed over each petri dish supported a watch glass containing 50% sucrose
solution and prevented the bees from direct contact with the aromatic oils.
* In this paper, "foragers" refer to the specially marked bees that were allowed to visit the watch glass
to collect sucrose solution.
 57

Training was begun on 29th January and continued daily throughout the test
period. The ten foragers selected for conditioning in each test were marked on the
dorsum of the thorax with individually distinctive enamel paint marks as described by
Wenner ( 1962). All other bees (recruits) arriving at the training stations on that day
were caught with forceps and killed in a flask of 70% ethyl alcohol. One observer
worked at each training station during the entire period the sucrose solution was
available, usually 2- 4 hours per afternoon.
During the first two tests the Caucasian bees were conditioned to geraniol, the
Cordovan bees were conditioned to citra!, and the Italian bees were allowed to forage
outside the cage (to prevent unnecessary interference from them). On 13th February,
the conditioning of the two colonies was reversed. The foragers from the Cordovan
colony were conditioned from citra! to geraniol without difficulty; the Caucasian bees
switched only slowly from geraniol to citra!, and many continued to visit the training
station scented with geraniol, even though this training station had been used previous-
ly by Cordovan foragers. Also, on this date, bees from the Italian colony were first
permitted to fly within the cage, and these foragers were conditioned to collect sucrose
solution at an unscented training station near the east wall of the cage (Fig. 1). Prior
to the last test the Italian bees were conditioned to a combination of citra! and
geraniol.
The searching activity of recruits from the colonies was evaluated as soon as 10
foragers were making regular visits to each training station. This was done by
arranging 16 petri dishes, containing a filter paper disc (as in training) with citra! or
geraniol or a mixture of them, or with no scent (control), 0·4 m apart in a 4 x 4 Latin
square on the ground at the centre of the cage (Fig. 1). However, no watch glasses
of sucrose were placed on the wire screens. Any bees landing on or hovering for
more than 5 seconds over a dish in the Latin square were counted as visitors, and
their colour (colony) was recorded. Such data were collected for 1 - It hours on
the afternoons of 9th, lOth, 17th and 19th February. The 10 conditioned foragers
were fed at the designated station every day; also, during the tests, they were allowed
to visit the training station. None visited the "wrong" station. They showed no
tendency to visit the Latin square except when the sucrose solution at the training
station was intentionally allowed to become depleted after the tests were finished at
the end of the day-to check whether the foragers would visit the Latin square. Since
many unmarked recruits were killed at the training stations during each feeding
period (after they had visited the Latin square and received no reward), this procedure
provided a continuous supply of new recruits for the tests.

Results
During the tests on 9th and lOth February, the Italian bees were left to forage outside
the cage (to prevent unnecessary interference). On both dates, most of the Caucasian
and Cordovan recruits visited the odour to which the ten foragers from their respective
colonies had been conditioned; however, many also visited the double-scented dishes
(Table lA, lB). Few recruits visited either the unscented control dishes or the dishes
with the scent to which bees from the other colony had been conditioned.
On 17th February, after the Cordovan and Caucasian foragers had been retrained
to geraniol and citra!, respectively, and the Italian foragers had been trained to visit an
unscented dish of sucrose, recruits were again tested with the Latin square arrangement
of scented and unscented dishes. Table 1C shows that the majority of Cordovan and
Caucasian bees visited the dishes with the scent to which they had been trained, a very
58

TABLE l. Visits by honeybee recruits to a 4 X 4 Latin square arrangement of petri dishes scented
with citra! (C), or geraniol (G), or citra! + geraniol (C + G), or control with no scent (0),
February 1970.

No. visits to dishes with

different scent(s)

Time Temperature Training

Date (MST) (oC) Bees Scent c G C+G 0

A 9 Feb. 15·20-16·20 24 Caucasian G 0 128 68 3

Cordovan c 94 0 42 1
B 10 Feb. 15·00-16·15 29 Caucasian G 4 40 14 I
Cordovan c 64 I 17 I
C 17 Feb. 14·50-15·50 31 Caucasian c 87 5 27 9
Italian 0 3 2 0 I
Cordovan G 3 99 55 4
D 19 Feb. 14·00-15·00 22 Caucasian c 98 2 15 2
Italian C+G 22 40 136 3
Cordovan G 2 68 44 I

few went to the other scent, and between approximately a fifth and a third of the
total visits were to dishes with the combined scent. Thus, these two types of bees
responded similarly when the training scents were reversed. Few of the Italian bees
visited any dishes in the Latin square, though they were foraging well at the unscented
training station during the test. Thus, under the conditions of this test, citra! and
geraniol exposed separately or in combination did not attract the unconditioned
Itali an bees.
Two days later, on 19th February, after Italian foragers had been conditioned to
citra! + geraniol at their training station, a large number of Italian recruits visited
double-scented dishes at the test-site, and smaller numbers went to dishes scented with
citra! alone or with geraniol alone (Table lD). As before, the Caucasian and
Cordovan foragers conditioned to dishes scented with citra! and with geraniol,
respectively, recruited bees to dishes that were scented with their training scent alone
and, to a lesser extent, to dishes scented with citra! + geraniol. In other words,
recruits from only two colonies were attracted to single-scented dishes, but recruits
from all three colonies were attracted to the double-scented dishes.

Discussion
If the results given in Table 1 are re-arranged to show the number of Cordovan and
Caucasian recruits that visited the single-scented dishes containing the scent to which
they were conditioned ("correct" response), and the numbers making an "incorrect"
response, the effect of the conditioning becomes even clearer (Table 2).
In the first two experiments (Caucasian bees conditioned to geraniol and Cordovan
bees conditioned to citra!), 68% of the total number of recruits visited dishes contain-
ing the single scent to which they were conditioned, and 30% went to double-scented
dishes. In the last two experiments (conditioning reversed) the results were very
similar. Only a small proportion of the total number visited dishes with the un-
familiar scent, or unscented dishes. This gives considerable support to the hypothesis
that recruits went to the double-scented dishes despite the presence of an unfamiliar
scent, and that they were not more attracted by the two scents than by the single one
to which the colonies had been conditioned.
 59

TABLE 2. Analysis of the effect of conditioning foragers to a single scent on the number of recruits
to single- and double-scented dishes (Caucasian and Cordovan bees only); data abstracted from
Tabk I.

Response: ''Correct" "Incorrect"

Training Double Unfamiliar

Date scent scent scent Control Totals

9 Feb. 222 110 0 4

10 Feb. 104 31 5 2
Total 326 (68·2%) 141 (29·5 ~lo) 5 (1·0%) 6 (1·3%) 478

17 Feb. 186 82 8 13
19 Feb. 166 59 4 3
Total 352 (67-6%) 141 (27·1 /~) 12 (2·3/~) 16 (3·1 ~';,) 521

Grand total 678 (67·9 %) 282 (28·2 %) 17 (I ·7 %) 22 (2·2%) 999

In the last test, 30% of the Italian recruits (conditioned to citra! + geraniol) were
attracted to either citra! alone or geraniol alone. This agrees with the conclusion
made by Gubin (1957) that bees trained to mixed scents can differentiate between
components and respond to each of them separately.
The results of this experiment indicate that recruits might be misdirected to another
plant species when two plants have in common the same chemical as a major com-
ponent of their flower aromas. Such a phenomenon would lead to the discovery of
new sources of pollen and nectar, and thus provide the colony with a diversity of
pollen and nectar.

Acknowledgements
I thank S. Taber, III, of this Laboratory for providing the Cordovan mutant Italian
bees; Dr. A. M. Wenner of the University of California at Santa Barbara for his
helpful suggestions and for reviewing the manuscript; and our technicians, J. Webster,
0. Ziehl, K. Laue and M. Cheeseman, for their help in training the bees and collecting
data.

References
FRISCH, K. VON (1967) The dance language and orientation of bees. Cambridge, Mass. : Harvard
University Press
GuBIN, V. A. (1957) [Sensitivity of the sense of smell in honeybees.] Pclzelovodslvo 34(7) : 17--19.
In Russian
JoHNSON, D. L. & WENNER, A.M. (1970) Recruitment efficiency in honeybees :studies on the role
of olfaction. J. apic. Res. 9(1) : 13-18
KoLTERMANN, R. (1969) Lern- und Vergessensprozesse bei der Honigbiene-aufgezeigt anhand von
Duftdressuren. Z. vergl. Physiol. 63(3) : 310-334
RIBBANDS, C. R. (1955) The scent perception of the honeybee. Proc. roy. Soc. B 143 : 367-379
WALLER, G. D. (1970) Attracting honeybees to alfalfa with citra!, geraniol and anise. J. apic. Res.
9(1) : 9-12
WENNER, A. M. (1962) Sound production during the waggle dance of the honey bee. Anim. Behav.
I 0(1/2) : 79-95