Behav Ecol Sociobiol (1999) 45: 95±106
ORIGINAL ARTICLE
Manfred Ayasse á Wolf Engels á Gunter LuÈbke
Timo Taghizadeh á Wittko Francke
Mating expenditures reduced via female sex pheromone modulation
in the primitively eusocial halictine bee, Lasioglossum (Evylaeus)
malachurum (Hymenoptera: Halictidae)
Received: 30 April 1998 / Accepted after revision: 24 July 1998
Abstract The present paper reports on behavioral ex-
periments and gas chromatographic analysis of chemical
communication in the mating biology of the primitively
eusocial sweat bee Lasioglossum (Evylaeus) malachurum.
In a dual-choice experiment, a female made odorless was
signi®cantly less attractive than an untreated one. At-
traction in L. (Evylaeus) malachurum is therefore medi-
ated by a female-produced sex pheromone. Further
bioassays showed that unmated gynes are more attrac-
tive to males than mated ones. Males are able to dif-
ferentiate between the two groups of females as little as
3 h after mating. Biotests with di€erent samples ob-
tained from attractive gynes showed surface extracts to
be most attractive. Behavioral tests with synthetic copies
of the compounds identi®ed as cuticular constituents of
virgin gynes were highly attractive to males; the volatile
bouquets consisting of n-alkanes, n-alkenes and iso-
pentenyl esters of unsaturated fatty acids were the most
attractive samples. Isopentenyl esters of unsaturated
fatty acids were the key compounds in inducing male
inspections as well as stimulating pounces and copula-
tory attempts. Virgin and nesting gynes di€ered clearly
in the relative and absolute amounts of the volatiles on
the cuticle. The total amount of volatiles was signi®-
cantly higher in virgin gynes and decreased in breeding
queens. Hydrocarbons were the dominant group of
M. Ayasse (&)
Institute of Zoology, Department of Evolutionary Biology
University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
e-mail: manfred.ayasse@univie.ac.at
Tel.: +43-1-313361398, Fax: +43-1-31336778
W. Engels
Institute of Zoology, Department of Developmental Biology
University of TuÈbingen, Auf der Morgenstelle 28
D-72076 TuÈbingen, Germany
G. LuÈbke, T. Taghizadeh, W. Francke
Institute of Organic Chemistry and Biochemistry
University of Hamburg, Martin-Luther-King-Platz 6
D-20146 Hamburg, Germany
Ó Springer-Verlag 1999
compounds in both groups of females. The relative
amounts of the wax-type ester, hexadecyl oleate, iso-
pentenyl esters, and a hitherto unidenti®ed steroid were
higher in attractive virgin gynes, while the relative pro-
portions of hydrocarbons and lactones dominated in
nesting queens. The site of sex pheromone production in
attractive young L. (Evylaeus) malachurum gynes re-
mains unknown. Head glands or Dufour's gland secre-
tions may be involved. Another possible source of the
`active principle' found among the cuticular lipids could
be glandular cells of the epidermis. The signi®cance of
modulation of female sex pheromone composition is
discussed in terms of a reduction in mating expenditures.
Key words Lasioglossum (Evylaeus) malachurum á
Sweat bee á Sex pheromone modulation á
Mating behavior á Chemical analyses
Introduction
Bees have evolved a remarkable variety of mating strate-
gies (Eickwort and Ginsberg 1980). Males may search for
mates at resources like nests or ¯owers (Alcock 1978;
Alcock et al. 1978). In social bees, the males may form leks
called drone congregations that function as rendezvous to
which gynes but not workers are attracted (Engels 1988).
A speci®c situation is seen in those ground-nesting bees
that can be found in aggregations of sometimes thousands
of nests (Michener 1990). In the sweat bee, Lasioglossum
(Evylaeus) malachurum, large numbers of reproductives
emerge during a short mating season (Knerer 1992). On
warm summer days, July through September in central
Europe (Westrich 1989), the nest aggregation area then
becomes a lek (Thornhill and Alcock 1983) with many
bees ¯ying around close to the ground. The daily opera-
tional sex ratio is strongly male biased (M. Ayasse, un-
published data), as in other bees (Wcislo 1992). Most
females are mated gynes and foraging workers and only
96
few are receptive virgins. Consequently, there is strong
competition among males for receptive females.
Within these ¯ying aggregations, males are able to
recognize conspeci®c gynes (Barrows 1975a). However,
only the sparse receptive gynes provoke excitement and
are approached. Among these, males prefer females to
which they are not related (Smith and Ayasse 1987).
Copulations are rarely seen and in L. (Evylaeus) ma-
lachurum, monandrous mating seems to be the rule
(Ayasse 1987; M. Ayasse and B.H. Smith, unpublished
data), although polyandry may occur (R.J. Paxton, un-
published data). Males can evidently di€erentiate gynes
(Ayasse 1990). In L. (Dialyctus) zephyrum this has been
interpreted as the consequence of an anti-aphrodisiac
label printed by the copulating male on the female, thus
making her unattractive for subsequent wooers (Kukuk
1985). Experiments on mating behavior, however, failed
to con®rm this assumption (Barrows et al. 1975; Wicslo
1987), although all these observations indicate that
males discriminate mates. Proximate requirements are
releaser signals functioning as discriminators and en-
abling the males to select promising mates. The relative
importance of visual and olfactory cues in this sweat bee
mating system remains an open question.
Previous studies on L. (Evylaeus) malachurum queens
have analyzed the content and ontogenetic patterns of
the volatile composition of Dufour's gland secretions
(Ayasse et al. 1990a). However, this gland turned out
not to be the main source of the female sex pheromone
(Ayasse et al. 1993). The question where the female sex
pheromone components are synthesized thus remained
open. Here we present evidence on the production site
and chemical composition of the signal emitted by re-
ceptive virgins. Analytical data were substantiated by
®eld bioassays. Modulation of the sex pheromone
composition apparently minimizes both molestation of
non-receptive gynes by males and ``useless'' mating at-
tempts. This considerably reduces mating expenditures
in the numerous encounters. In halictine bees, these
mating economics may ultimately compensate the con-
straints imposed by gregarious nesting and by the mat-
ing strategy described above.
The present paper reports on behavioral experiments
and gas chromatographic analysis of the volatile signal
used for chemical communication in the mating biology of
the primitively eusocial sweat bee L. (Evylaeus) malachu-
rum. The sex pheromone of the species was identi®ed by
behavioral tests and chemical analysis. The odor bou-
quets of unmated (attractive) gynes [de®ned following
Michener (1990) as females that will found nests or have
done so, but do not have worker associates] and breeding
(unattractive ) queens (females that have worker associ-
ates) were identi®ed and compared. The objective was to
answer the following questions. Do the males recognize
females' mating status? Which parts of the body emit the
sex pheromone and which glands produce it? An e€ort
was also made to investigate the possible function of dif-
ferent chemical classes of compounds in attracting males
and stimulating copulatory behavior.
Methods
Natural history
L. (Evylaeus) malachurum is a primitively eusocial sweat bee com-
mon throughout the west Palearctic region (Knerer 1992). Nesting
aggregations with up to several thousand nests can be found in bare
soil sometimes covered with scattered vegetation. Seasonal activity
starts in early spring (March±April) when nests are founded by
single overwintered gynes. The ®rst brood consists only of workers
(Sakagami 1974), although, depending on the climatic region, a few
males can occasionally be found (Knerer 1992). In the last brood of
the year (in TuÈbingen the second and in Vienna the third brood),
®rst males and several days later female reproductives emerge.
Mating takes place at the nesting site, where large numbers of
males patrol just above the nest entrances. Only young mated gynes
overwinter and start nesting in the following year.
Mating experiments were conducted from July to early Sep-
tember 1985±1993. Bees were collected at two nesting sites close to
TuÈbingen (south Germany) and one near MoÈdling (Austria).
Attractiveness of odorless and untreated gynes
The ®rst experiment was a control to compare the importance of
female odor versus visual cues in male attraction. Two to 3 days
before the experiment was performed, emerging gynes were collected
from the nest entrance early in the morning. Each gyne was placed in
a cooled, clean plastic cup (Eppendorf) and killed by freezing. Half
the females were extracted in pentane (Uvasol, Merck) for at least
24 h and subsequently heated at 60 °C for 8 h. This group of females
was almost free of volatiles and termed ``odorless.'' The other half
was stored in the freezer and termed ``untreated.'' The vials con-
taining both groups of females were brought to the ®eld in a con-
tainer with ice. Before each test, an odorless and an untreated gyne
were removed from the plastic cups and individually ®xed on insect
pins. In a dual-choice experiment lasting 3 min, the number of
contacts of patrolling males with the two females, pinned 1.5 cm
above the ground and 5 cm apart, was counted. The behavioral event
termed contact could either be a brief touch or a copulatory attempt.
Each pair of gynes was tested only once.
Attractiveness of mated and unmated young gynes
In a second experiment we investigated whether males are able to
discriminate between unmated and mated young gynes. In the ®eld,
nests were excavated to obtain gyne pupae. In the laboratory, single
pupae were transferred to 1.5 ml plastic cups (Eppendorf), which
were closed with a piece of moist ®lter paper. The cups were stored
in the dark at room temperature (21 °C), and every day a few drops
of water were added to the ®lter paper until gynes emerged from the
pupae.
For each dual-choice experiment, pairs of sister bees were used.
Three days after emergence, one of the two sister bees mated in a
¯ight cage (40 ´ 40 ´ 40 cm) with males collected in the ®eld. The
unmated gynes were ¯ying in a cage without males. Three hours after
mating, both sister bees (one unmated, one mated) were frozen and
stored in the freezer at ±20 °C until they were used in the ®eld tests on
the following day, where handling and testing were as described for
the choice experiment using odorless and untreated bees.
Mating experiments with odor extracts
and synthetic compounds
Mating experiments similar to those described by Smith and
Ayasse (1987) were performed to prove the role of volatiles in
attracting males and stimulating copulatory behavior. Natural

extracts and blends of synthetic copies of the volatiles identi®ed in
unmated young gynes were applied on pinned odorless gynes
(dummy) treated before each test as described below. In each
experiment, a dummy bee was impregnated with 10 ll of a pen-
tane solution containing one female equivalent of the test solu-
tion. Before the dummy bee was ®xed 1.5 cm above the ground,
the solvent was allowed to evaporate for 1 min. To avoid habit-
uation, the tests were alternately carried out at two or three dif-
ferent spots within the male patrolling area; these sites were at
least 10 m apart. Earlier marking experiments showed that the
males do not move freely throughout the nesting aggregation
(Smith and Ayasse 1987) and, therefore, di€erent males were
present at the three testing spots. Each odorless bee was used only
once per day; it was then extracted with pentane (Uvasol) and
dried in an oven for at least 8 h at 60 °C prior to renewed use.
Within a testing period of 3 min, three di€erent male reactions
were recorded (after Kullenberg 1973): inspection ± a patrolling
male approaches to within 5 cm of the dummy bee; pouncing ± a
male contacts the dummy bee with its head or antennae; copulation
± a male mounts a dummy bee and tries to copulate.
To combine the tests carried out on di€erent days and at di€erent
localities where the number of patrolling males was not the same, the
male reactions were standardized by measuring their ¯ight activity.
A 1-m-long wire was mounted at the experimental area. The number
of males crossing the wire in 3 min was counted at least every 30 min.
For each 3-min test, individual reactions of the males were divided by
the mean male ¯ight activity and multiplied by 100.
Origin of the female sex pheromone
In a ®rst experiment, attempts were made to localize the source of
the sex pheromone of young and attractive gynes (collected 1 day
before the experiments). Only females that were attractive to males
were used. This was checked by placing one female together with
three to ®ve males in a small plastic tube (10 cm high, diameter
5 cm). If a male tried to copulate within the next 30 s, the female
was killed by freezing and used for odor extractions as described
below. We checked for the emptiness of the spermatheca by dis-
sections and found all of the attractive females to be unmated.
The following samples were used in ®eld experiments:
(1) Surface extracts. Single frozen females were extracted for 15 s
in 1 ml diethyl ether (Merck). Subsequently, the diethyl ether
was evaporated in a glass capillary in a water bath (40 °C) and
10 ll of pentane was added.
(2) Extracts of whole females. The gynes were placed in 0.5 ml
pentane (Uvasol) and stored at room temperature (21 °C) for
24 h, before they were removed from the solvent.
(3) Extracts of the Dufour's glands. Individual Dufour's glands
were extracted in 100 ll pentane (Uvasol) for 24 h.
(4) Extracts of the head. After decapitation, heads were extracted
in 100 ll pentane (Uvasol) for 24 h.
Further extracts of whole females and heads were obtained from
attractive young gynes after rinsing with diethyl ether before the
females or heads were extracted in pentane. Prior to transporting
the test solutions to the ®eld, they were concentrated and adjusted
to 10 ll/female equivalent in a water bath at 40 °C.
Biotest with synthetic copies of the compounds
identi®ed in surface extracts
To examine the possible behavior-releasing e€ect of compounds
identi®ed in the surface extracts of virgin young gynes in attracting
males and releasing copulatory behavior, synthetic copies of the
volatiles were used in another series of tests. The biological activity
of a total of 33 compounds was tested. Mixtures of synthetic copies
were prepared according to the results of GC-MS analysis of sur-
face extracts (Ayasse 1991). The relative and absolute amounts of
the compounds in all test solutions were checked by GC. The total
mixture contained n-alkanes, n-alkenes, saturated and unsaturated
97
macrocyclic lactones, ethyl esters, as well as isopentenyl esters of
saturated and unsaturated fatty acids. A list of all synthetic com-
pounds and mixtures is provided in Table 1. As control, 10 ll
pentane was used (Uvasol). In the ®eld, test solutions were added
onto odorless ``dummy bees.'' All tests were performed in the same
manner as the tests with gland extracts or surface extracts.
Chemical analysis
The surface extracts of virgin young gynes and old breeding
queens were analyzed on a Carlo Erba Fractovap 2450 gas
chromatograph, equipped with a DB5 capillary column
(30 m ´ 0.32 mm internal diamter), operating at 120 °C for 30 s,
followed by programming to 280 °C at 4 °/min. For quantitative
analyses, n-octacosane was added as an internal standard. Re-
sponse factors were determined by single-level calibration.
Structure elucidation of individual compounds was based on GC-
MS (VG70/250 SE instrument, Vacuum Generators, Manchester,
UK, linked to a HP 5890; gas chromatographic condition as
mentioned above) and comparison with mass spectra reported in
the literature (Mc La€erty and Stau€er 1989), and comparison of
gas chromatographic retention times (co-injection) with those of
authentic reference samples. Double-bond positions in unsatu-
rated compounds were assigned according to Buser et al. (1983)
and Dunkelblum et al. (1985). Determination of the stereo-
chemistry of double bonds was achieved by comparison of re-
tention times using corresponding reference samples including
DMDS derivatives. The erythro- and threo-aducts can be well
separated by gas chromatography.
Statistics
Di€erences in reactions recorded in the choice experiments for
unmated and mated females and odorless and untreated females
were examined with a Wilcoxon signed-ranks test (Sokal and Rohlf
1995). Di€erences in numbers of inspections, pounces and copu-
latory events of all testing solutions in comparison to the control
(pentane blank) were examined with a Mann-Whitney U-test (So-
kal and Rohlf 1995). The relative and absolute amounts of single
compounds in virgin gynes and nesting queens were compared by a
Mann-Whitney U-test (Sokal and Rohlf 1995).
Results
L. (Evlaeus) malachurum gyne odors as sexual releasers
In the ®rst choice experiment, where the attractiveness of
an odorless female was compared with that of an un-
treated one, the latter received signi®cantly more copu-
lation attempts during the 3-min testing period (Fig. 1;
P < 0.01, Wilcoxon signed-ranks test). As the attrac-
tiveness of the females was highest during the 1st minute
and decreased in the 2nd and 3rd minute, some kind of
learning or habituation might have taken place. The
results demonstrate that attraction in L. (Evylaeus) ma-
lachurum is mediated by a female-produced sex phero-
mone.
Attractiveness of unmated and mated young gynes
Unmated and mated young gynes also di€ered in their
attractiveness to the males. Unmated gynes received a
signi®cantly higher number of pounces and copulatory
98

Table 1 Synthetic copies of the

compounds identi®ed in the Compounds Amount Mixtures of synthetic compounds
surface extracts of attractive (ng)
young (Evylaeus) malachurum 1 2 3 4 5 6 7 8 9
gynes. Between August 1989
and 1993 the following volatile Alkanes and alkenes
mixtures were tested in the ®eld: Heptadecane 3.27 x x x
1 total mixture 1989, 2 total Nonadecane 5.73 x x x
mixture 1990, 3 alkanes, 4 al- Heneicosane 2.78 x x x
kenes, 5 saturated and un- Tricosane 157.26 x x x
saturated lactones, 6 saturated Pentacosane 190.83 x x x
lactones, 7 ethyl esters and iso- Heptacosane 165.89 x x x
pentenyl esters of saturated Nonacosane 124.60 x x x
fatty acids, 8 isopentenyl esters Triacontane 81.98 x x x
of saturated and unsaturated (Z)-7-Heneicosene 2.76 x x x
fatty acids, 9 isopentenyl esters (Z)-7-Tricosene 2.32 x x x
of unsaturated fatty acids. Re- (Z)-9-Pentacosene 34.86 x x x
lative and absolute amounts of (Z)-9-Heptacosene 99.66 x x x
single compounds in the mix- (Z)-7-Heptacosene 1.95 x x x
tures were the same as found in (Z)-9-Nonacosene 126.19 x x x
gas chromatographic analyses Lactones
of surface extracts of virgin 18-Octadecanolide 28.55 x x x x
queens (Ayasse 1991) 20-Eicosanolide 16.44 x x x x
22-Docosanolide 27.71 x x x x
18-Octadec-(Z)-9/-(Z)-11-enolide 13.82 x x
20-Eicos-(Z)-11/-(Z)-13-enolide 26.78 x x
Isopentenyl esters
3-Methyl-3-butenyl octadecanoate 48.10 x x x x
3-Methyl-2-butenyl octadecanoate 5.54 x x x x
3-Methyl-3-butenyl eicosanoate 16.92 x x x x
3-Methyl-3-butenyl docosanoate 23.11 x x x x
3-Methyl-2-butenyl docosanoate 1.10 x x x x
3-Methyl-3-butenyl tetracosanoate 66.17 x x x x
3-Methyl-2-butenyl tetracosanoate 38.38 x x x x
3-Methyl-3-butenyl linoleate 4.86 x x x
3-Methyl-3-butenyl oleate 12.59 x x x
3-Methyl-3-butenyl linolenoate 2.92 x x x
3-Methyl-3-butenyl eicos-11-enoate 0.43 x x x
Ethyl esters
Ethyl octadecanoate 7.26 x x x
Ethyl eicosanoate 5.12 x x x
Ethyl docosanoate 4.62 x x x

attempts compared to mated ones (Fig. 2, P < 0.05,
Wilcoxon signed-ranks test). Males are already able to
discriminate between both groups of females 3 h after
mating. Corresponding to the results of the ®rst exper-
iment, the number of male reactions decreased in the
2nd and 3rd minute after the females were o€ered to the
males.
Source of the female sex pheromone
Biotests with di€erent samples obtained from attractive
gynes showed the surface extracts (SE) to be the most
attractive principle (Fig. 3, Table 2). Fewer male reac-
tions were recorded when dummy bees were impreg-
nated with extracts of whole females (WAE) or heads
(HE). Extracts of Dufour's glands (DGE) and also those
produced from heads after surface extraction (HE a. se.)
elicited the lowest number of male reactions. A dummy
bee impregnated solely with pentane (control) was sig-
ni®cantly less attractive than a dummy bee to which one
of the volatile mixtures of attractive young gynes was
added. The attractiveness of odorless bees to the males
may be due to visual cues. Only approaches or short
contacts with the antennae of the male were observed,
never copulatory attempts.
The surface extracts were found to be the most at-
tractive sample and yielded the same number of male
reactions as frozen virgin gynes (Ayasse 1987; Smith
and Ayasse 1987): the male reactions here were there-
fore set at 100%. Referring to this, more than 70% of
the males were attracted to a dummy bee impregnated
with Dufour's gland, head or whole-body extracts.
Surface extracts, whole-animal and head extracts elic-
ited a signi®cantly higher number of pouncing reactions
than the pentane blank (Table 2). The result was almost
the same when the number of copulatory attempts were
considered: surface extracts applied to odorless dummy
bees released signi®cantly more copulatory attempts
than the control (pentane blank, Fig. 3, Table 2). Since
among the other samples tested in the ®eld, only ex-
tracts from whole animals and from the cuticle surface
stimulated signi®cantly more copulatory attempts than
the control, males are obviously stimulated to mate

with a female by the compounds covering the cuticular
surface.
The number of pouncing events was the same in ex-
periments with whole female extracts after extraction of
the cuticular surface (WAE a. se.) and head extracts
(HE); furthermore, the copulatory events recorded with
whole female extracts (WAE a. se.) and Dufour's gland
secretions were also the same (Fig. 3). Therefore, vola-
tiles of the head seem to be important in attracting males
from a longer distance and eliciting pouncing events,
while Dufour's gland secretions might stimulate copu-
latory attempts. As more copulatory events were re-
corded using whole female extracts (WAE) than
Dufour's gland secretions, other glands or glandular
cells seem to be important as well.
Behavior experiments with synthetic compounds
Synthetic copies of the compounds identi®ed on the
cuticular surface of virgin gynes were highly attractive to
males (Table 3). Mating experiments showed the total
mixtures and the volatile bouquets consisting of alkanes,
alkenes and isopentenyl esters of unsaturated fatty acids
to be the most attractive samples (Fig. 4, Table 3). In
tests with all these mixtures, the number of inspecting
males was signi®cantly higher than in the control. Hy-
drocarbons, isopentenyl esters of saturated and unsatu-
rated fatty acids, and the total mixtures of synthetic
volatiles yielded male inspections at the 80% level
compared to the surface extracts. Isopentenyl esters of
unsaturated fatty acids were found to be the most im-
portant compounds in stimulating pouncing events
(50% compared with the surface extracts). Less stimu-
lating but still more reactive than the control were the
total mixture of volatiles tested in 1990 and isopentenyl
Fig. 1 Comparison of the mean male response to untreated and
odorless L. (Evylaeus) malachurum gynes over a 3-min testing
period. In a total of 16 tests, over three successive 1-min intervals,
odorless queens were signi®cantly less attractive than the untreated
ones (Wilcoxon signed-ranks test, P < 0.01) (vertical bars SE of
the mean)
99
esters of saturated and unsaturated fatty acids (25%
compared with the surface extracts).
Among all synthetic mixtures, isopentenyl esters of
unsaturated fatty acids stimulated the highest number of
copulatory attempts, although only four of nine identi-
®ed compounds were used. All other mixtures contain-
ing isopentenyl esters of unsaturated fatty acids
triggered copulatory attempts, as did a mixture of sat-
urated and unsaturated lactones. A striking result was
the lower number of reactions when other compounds
were included beside the isopentenyl esters of unsatu-
rated and saturated fatty acids. This may be caused by a
potentially higher rate of error when mixing a high
number of compounds. Synthetics released only 10% of
the copulatory attempts induced by dummy bees im-
pregnated with cuticular surface washings.
Chemical analysis
Overall, 131 compounds were identi®ed in the surface
extracts [for a complete list of compounds see Ayasse
(1991) and Taghizadeh (1996)]. The mixture was made
of straight-chain saturated and unsaturated hydrocar-
bons with 11±33 carbons (alkanes, alkenes, alkadienes),
methyl branched hydrocarbons, carboxylic acids, alco-
hols, various types of esters, saturated and unsaturated
lactones, and compounds belonging to other chemical
classes.
Virgin and nesting gynes di€ered clearly in the rela-
tive and absolute amounts of the volatiles identi®ed on
the cuticular surface (Table 4, Fig. 5). Hydrocarbons
and a long-chain wax-type ester hexadecyl oleate rep-
resent the main compounds in both groups of females.
In breeding queens, hydrocarbons formed the dominant
group of compounds. The total amount of volatiles was
Fig. 2 Comparison of the mean male response to unmated (virgin)
and mated young L. (Evylaeus) malachurum gynes. In 14 dual-
choice experiments, mated queens were signi®cantly less attractive
to the males than unmated ones as early as 3 h after copulation
(Wilcoxon signed-ranks test, P < 0.05) (vertical bars SE of the
mean)
100

Discussion

Are chemical cues important in mate recognition by L.

(Evylaeus) malachurum males?

In L. (Evylaeus) malachurum, queens nest in dense ag-

Fig. 3 Attractiveness of di€erent odor samples of virgin young
gynes to the males. Within a testing period of 3 min, three di€erent
male reactions were recorded (after Kullenberg 1973): inspection a
patrolling male approached to within 5 cm of the dummy bee;
pouncing a male contacted the dummy bee with its head or
antennae; copulation a male mounted a dummy bee and tried to
copulate. To combine the tests carried out at di€erent days and
localities where the number of patrolling males was not the same,
male reactions were standardized by measuring the ¯ight activity.
A 1-m-long wire was mounted at the experimental sites. The
number of males crossing the wire over a 3-min period was counted
at least every 30 min. For each 3-min test, individual reactions of
the males were divided by the mean male ¯ight activity and
multiplied by 100. Since surface extracts elicited the same number
of male reactions as did living virgin queens, the mean coecient of
attractiveness of the three registered male reactions was set at 100%
(statistics in Table 2). All investigated odor samples (for abbre-
viations see Table 2) were more attractive to the males than the
control (pentane blank)
signi®cantly higher in virgin gynes and decreased in
breeding queens (virgin: x ˆ 8.55 lg, breeding: x ˆ
3.36 lg; Mann-Whitney U-test P < 0.05). The relative
amounts of hexadecyl oleate, isopentenyl esters and a
steroid were higher in attractive virgin gynes, while the
relative proportions of hydrocarbons and lactones were
higher in nesting ones.
gregations and remain receptive for only a short time
after emergence (StoÈckhert 1923). Therefore, there is a
strong selective pressure for a male to recognize a virgin
female before the huge number of other conspeci®c
males. In fact, virgin young gynes, willing to mate, re-
leased a higher intensity of the typical search ¯ights in
the males, even before they appeared at the nest entrance
(M. Ayasse, unpublished observations). A typical
chemotactically mediated searching behavior can be
observed. Males assembled closely around nesting areas,
where virgin gynes soon appeared. Some even inspected
entrances of nests containing receptive gynes. Other
males on the ground started to clean their antennae with
the front legs, a behavior indicating sexual stimulation
by the gyne odor.
To minimize the time and energy required to resist
male mating attempts, already mated queens may release
unambiguous signals of non-receptivity (Alcock et al.
1978). Unlike related species such as L. (Dialictus)
zephyrum (Barrows 1975a; Wcislo 1987), L. (Evylaeus)
malachurum males were never observed pouncing on
black spots, stones, other insects and worker females
while they perceive the odor of a virgin female. This
con®rms the relevance of pheromonal interactions. Di-
rect evidence came from our ®eld bioassay with dead,
pinned queens made odorless by solvent extraction: such
dummies were widely ignored by the males, in contrast
to untreated gynes, which were objects of frequent
copulation attempts. In L. (Evylaeus) malachurum, ol-
factory cues clearly provide information for both males
and female sexuals; this minimizes both the disturbance
of already mated gynes and time-consuming mating at-
tempts by males.

Table 2 Attractiveness of di€erent extracts obtained from unmated, attractive young gynes for L. (Evylaeus) malachurum males in the
®eld. (Coecient of attractiveness number of male reactions per 3 min ´ 100/¯ight activity, x mean, Sx SE of mean)

Test solution (n) Coecient of attractiveness

Inspection Pouncing Copulation

x Sx x Sx x Sx

Surface extracts (SE) (37) 83.33 5.51** 20.79 5.28** 2.98 1.21**
Whole-animal extract (WAE) (20) 83.11 11.56** 9.67 1.96** 1.28 0.70**
WAE after surface extraction (WAE a. se.) (23) 80.90 10.52** 5.65 1.88 0.09 0.09
Dufour's gland extract (DGE) (22) 72.82 7.90** 2.49 0.61 0.08 0.08
Head extract (HE) (7) 80.78 8.38** 5.61 3.48* 0.00 0.00
HE after surface extraction (HE a. se.) (18) 61.17 8.32* 5.18 1.64* 0.00 0.00
Control (pentane blank) (58) 37.59 3.11 1.98 0.40 0.00 0.00

*P < 0.05; **P < 0.001 relative to control in Mann-Whitney U-test

 101

Table 3 Attractiveness of mixtures (see Table 1) of synthetic copies number of male reactions per 3 min ´ 100/¯ight activity, x mean,
of the compounds identi®ed in surface extracts of attractive young Sx SE of mean). Mixtures of isopentenyl esters of unsaturated fatty
L. (Evylaeus) malachurum gynes. (Coecient of attractiveness acids were found to be most attractive to the males

Test solution (n) Coecient of attractiveness

Inspection Pouncing Copulation

x Sx x Sx x Sx

(1) Total mixture tested 1989 (22) 78.96 9.22** 1.99 0.63 0.00 0.00
(2) Total mixture tested 1990 (20) 69.92 5.11** 4.99 0.90** 0.11 0.11
(3) Alkanes (23) 72.10 8.59** 2.82 0.79 0.00 0.00
(4) Alkenes (20) 72.93 13.07** 3.88 1.66 0.00 0.00
(5) Saturated and unsaturated lactones (20) 57.29 4.65** 3.02 0.93 0.10 0.10
(6) Saturated lactones (20) 58.44 6.03** 4.85 1.50* 0.00 0.00
(7) Isopentenyl esters of saturated fatty acids+ethyl esters (22) 48.83 5.03 3.03 0.70 0.00 0.00
(8) Isopentenyl esters of saturated and unsaturated fatty acids (19) 64.83 5.74** 5.53 1.55** 0.05 0.05
(9) Isopentenyl esters of unsaturated fatty acids (19) 77.97 8.71** 10.69 2.14** 0.27 0.18*
(10) Control (pentane blank) (58) 37.59 3.11 1.98 0.40 0.00 0.00
*P < 0.05; **P < 0.001 relative to control in Mann-Whitney U-test

mated young gynes may be an anti-aphrodisiac that

Fig. 4 The biological activity of mixtures of synthetic copies of
surface extracts. The relative and absolute amounts of the
compounds in all test solutions were prepared according to the
results of GC-MS analysis of surface extracts (Table 4). The total
mixture contains alkanes, alkenes, saturated and unsaturated
macrocyclic lactones, ethyl esters, as well as isopentenyl esters of
saturated and unsaturated fatty acids. A list of all synthetic
compounds and mixtures is shown in Table 1. For a control, 10 ll
of pentane blank (Uvasol, Merck) was used. In the ®eld, test
solutions were added onto odorless ``dummy bees.'' All tests were
performed in accordance with the tests with females and gland
extracts or surface extracts. The odor mixture of isopentenyl esters
of unsaturated fatty acids was most attractive for males and
released copulatory attempts (statistics in Table 3)
Are chasing males capable of discerning
receptive young gynes?
Male competition for receptive females is evidently se-
vere in lek situations and can be observed in many bee
species (see Alcock et al. 1978; Thornhill and Alcock
1983); this should create selection for abilities to rapidly
habituate to cues associated with unreceptive females. In
fact, as early as 3 h after copulation took place,
L. (Evylaeus) malachurum males were able to discrimi-
nate unmated and mated young gynes (Fig. 2) by odor
cues. An explanation for the lower attractiveness of
males apply on a female during copulation. Other males
possibly perceive this anti-aphrodisiac and avoid wast-
ing time copulating with an already mated and therefore
unreceptive female. At the same time, the marking male
could increase its ®tness, since all of the diploid eggs a
female produces would be fertilized by his sperm. Based
on behavioral experiments in L. (Dialictus) zephrum,
Kukuk (1985) proposed this so-called anti-aphrodisiac
hypothesis, which was, however, not veri®ed by subse-
quent studies with the same species (Wicslo 1987, 1992).
An alternative explanation for the lower attractive-
ness of young gynes after mating could be a post-cop-
ulatory change in the female's pheromonal signal,
induced by mating. Several arguments support this as-
sumption. First, our ®ndings show that in young
L. (Evylaeus) malachurum gynes, the relative propor-
tions and absolute amounts of volatiles of extracts of the
Dufour's gland, of the head and also of surface extracts
changed after mating (Ayasse 1991; Ayasse et al. 1991,
1993). Furthermore, the total amount of volatiles on the
cuticular surface decreases. Therefore, we can exclude an
anti-aphrodisiac on the cuticular surface of recently
mated young gynes. For instance, here we present evi-
dence that isopentenyl esters, which were found to be the
most attractive compounds of virgin gynes, almost dis-
appeared within a few hours after mating (Fig. 5). Dif-
ferences in volatile constituents of virgin and mated
females have been shown in several bee species by be-
havior experiments (Wcislo 1992; Engels et al. 1993;
Dutzler and Ayasse 1996) and by chemical analyses
(Engels and Engels 1988; Ayasse et al. 1990a,b ; Engels
et al. 1993, 1997). A mating-induced termination of fe-
male sex pheromone production has been shown in
cockroaches (Schal et al. 1997), moth (Raina 1997),
lepidoptera (McNeil et al. 1997), and in other insects. In
Heliothis zea, the male accessory glands have been im-
plicated as the source of a peptide that terminates
pheromone production in mated females. This peptide is
transferred to the female at the time of mating (Raina
102

Table 4 Relative amount (%) of

volatile compounds identi®ed in Compound Relative amount (%)
surface extracts of virgin (n ˆ 7)
and nesting (n ˆ 12) L. (Evylaeus) Unmated Nesting
malachurum queens (x mean, Sx
SE of mean) x Sx x Sx

Alkanes
Heptadecane 0.10 0.06 0.00* 0.00
Nonadecane 0.06 0.02 0.01* 0.01
Heneicosane 0.13 0.03 0.37* 0.11
Tricosane 6.46 1.51 19.87** 1.55
Pentacosane 6.79 1.29 4.74* 0.50
Heptacosane 6.47 1.09 15.16* 1.37
Nonacosane 5.24 0.94 12.11* 1.60
Triacontane 3.42 1.02 1.46 0.46
Alkenes
(Z)-7-Heneicosene 0.12 0.08 0.00 0.00
(Z)-9-Tricosene 0.00 0.00 0.15* 0.03
(Z)-7-Tricosene 0.13 0.09 0.36* 0.07
(Z)-9-Pentacosene 1.50 0.85 4.82* 1.08
(Z)-7-Pentacosene 0.00 0.00 0.25** 0.06
(Z)-9-Heptacosene 3.31 0.84 5.65* 0.59
(Z)-7-Heptacosene 0.10 0.07 0.96** 0.08
(Z)-9-Nonacosene 6.07 1.69 6.56 0.61
Lactones
18-Octadecanolide 1.36 0.66 1.58 0.49
20-Eicosanolide 0.86 0.33 5.21* 1.90
22-Docosanolide 0.87 0.46 3.75** 1.07
24-Tetracosanolide 0.00 0.00 0.35* 0.13
18-Octadec-(Z)-9/(Z)-11-enolidea 0.61 0.17 0.27 0.11
20-Eicos-(Z)-11/(Z)-13-enolidea 1.22 0.28 1.92 0.49
22-Docos-(Z)-13/(Z)-15-enolidea 1.56 0.43 1.82 0.33
24-Tetracos-(Z)-15/(Z)-17-enolidea 0.07 0.04 0.08 0.03
Isopentenyl esters
3-Methyl-3-butenyl octadecanoate 2.29 1.22 0.09* 0.05
3-Methyl-2-butenyl octadecanoate 0.23 0.11 0.02 0.01
3-Methyl-3-butenyl eicosanoate 0.78 0.27 0.09** 0.05
3-Methyl-3-butenyl docosanoate 1.11 0.34 0.03** 0.02
3-Methyl-2-butenyl docosanoate 0.05 0.05 0.05 0.03
3-Methyl-3-butenyl tetracosanoate 2.39 0.67 0.00** 0.00
3-Methyl-2-butenyl tetracosanoate 1.38 0.49 0.20* 0.06
3-Methyl-3-butenyl linoleate 0.24 0.12 0.07 0.03
3-Methyl-3-butenyl oleate 0.63 0.42 0.01* 0.01
3-Methyl-3-butenyl linolenoate 0.19 0.10 0.02 0.02
3-Methyl-3-butenyl eicosadienoateb 0.16 0.09 0.06 0.02
3-Methyl-3-butenyl eicosenoatec 0.02 0.02 0.00 0.00
3-Methyl-3-butenyl eicosatrienoateb 0.12 0.07 0.00 0.00
Ethyl esters
Ethyl octadecanoate 0.42 0.11 0.28 0.04
Ethyl eicosanoate 0.26 0.08 0.21 0.02
Ethyl docosanoate 0.18 0.05 0.40 0.04
Ethyl oleate 0.35 0.08 0.01** 0.01
Ethyl linoleate 1.16 0.22 0.03** 0.01
Ethyl eicosenoateb 0.17 0.09 0.54* 0.10
Ethyl docosenoateb 0.54 0.11 0.46 0.05
Carboxylic acids
Hexadecanoic acid 0.50 0.50 0.38 0.12
Hexadecenoic acidb 0.00 0.00 0.10 0.07
9-Octadecenoic acid 2.87 1.09 0.01* 0.01
Eicosenoic acidb 0.42 0.23 0.72 0.20
Docosenoic acidb 0.18 0.08 2.55** 0.34
Tetracosanoic acid 0.53 0.09 1.58** 0.27
Other compounds
Unknown (M=326) 0.00 0.00 2.55 0.79
Unknown (M=354) 0.00 0.00 2.31* 0.84
Steroid 9.33 2.64 2.55 0.34
Hexadecyl oleate 27.43 11.39 0.39** 0.17
*P < 0.05; **P < 0.001 in Mann-Whitney U-test
a
Unsaturated lactones are mixtures of two positional isomers
b
Position of the double bond has not been determined
c
Mixture of two positional isomers (11, 13)

Fig. 5 Comparison of the relative amounts of n-alkanes (1),
n-alkenes (2), a wax-type ester (hexadecyl oleate, 3), saturated
macrocyclic lactones (4), unsaturated macrocyclic lactones (5),
isopentenyl esters of saturated (6) and unsaturated (7) fatty acids,
ethyl esters saturated (8) and unsaturated (9), fatty acids (10) and a
steroid compound (11) in surface extracts of young virgin and old
nesting L. (Evylaeus) malachurum queens (Mann-Whitney U-test:
*P < 0.05; **P < 0.001)
1989). Whether a similar mechanism is responsible for
the post-copulatory change of the pheromonal signal in
L. (Evylaeus) malachurum remains open.
Where is the female sex pheromone located
and where is it produced?
In halictid bees, the Dufour's gland takes up a large part
of the abdomen. Because of its large size, many inves-
tigations on pheromonal communication have focused
exclusively on the contents of this gland (Dueld et al.
1984). In L. (Dialictus) zephrum, Smith et al. (1985)
found that macrocyclic lactones of the Dufour's gland
are used as recognition signals and sex pheromones.
However, their experiments failed to compare the at-
tractiveness of volatiles from the Dufour's gland and
other possible sources like the head and the cuticle sur-
face. Furthermore, only short contacts of the males and
no attempts to copulate were recorded. In our experi-
ments, extracts of the Dufour's gland and the head were
less attractive than the volatiles on the cuticular surface,
which released the highest intensity of male mating be-
havior (Fig. 3). In L. (Dialictus) zephyrum, compounds
on the cuticular surface were also important in attracting
males (Barrows 1975b; Wisclo 1987).
Since dummies impregnated with cuticular extracts of
virgin gynes released the same number of male reactions
as did frozen receptive gynes (Ayasse 1987) and, on the
other hand, the attractiveness of dead and active Las-
ioglossum gynes are similar (Barrows 1975b; Smith
1983), the female sex pheromone in sweat bees is
103
evidently located on the cuticular surface, and probably
distributed all over the body.
The production site of the sex pheromone in
L. (Evylaeus) malachurum gynes remains unknown.
Secretions from cephalic glands and/or the Dufour's
gland may be involved. Another possible source of the
`active principle' on the cuticle could be glandular cells
of the epidermis below tergites 3±5 or from the sternite 6
described by Altenkirch (1962) in several species of the
genus Halictus. In Eucera palestinae, secretions of tergal
glands released mating behavior in the males (Shimron
and Hefetz 1985).
The volatile bouquet on the cuticle could be a mix-
ture of compounds produced in several glands. Our
®ndings that all test solutions released signi®cantly
more male reactions than the control support this hy-
pothesis. The speci®city of the volatile bouquet on the
cuticle of attractive young gynes may be a result of
combining the secretions from di€erent glands, and a
quick change of the bouquet after mating might be
e€ected by ceasing production or terminating secretion
of compounds.
Which cuticular volatiles of a gyne
attract males most e€ectively?
The present behavior experiments with synthetic cutic-
ular volatiles demonstrated a contribution of iso-
pentenyl esters to the female sex pheromone in
L. (Evylaeus) malachurum. When a test mixture con-
tained such compounds, the males not only inspected
dummy bees but also tried to copulate. Although
isopentenyl esters have also been found in other Halic-
tinae (Dueld et al. 1981, 1984; Smith et al. 1985; Hefetz
et al. 1986) and in Nomiinae (Dueld et al. 1982), their
potential role in chemocommunication remained un-
known. In L. (Evylaeus) malachurum, the prevalence of
isopentenyl esters is a caste-speci®c trait of gynes, and
the Dufour's gland bouquet di€ers in young gynes and
young workers (Ayasse et al. 1993). A more complete
copy of the virgin gyne pheromone containing more of
the identi®ed compounds might result in male reaction
intensities similar to those released by a live bee, because
the behavioral reactions released by a complete mixture
of compounds is often higher than the sum of the
behavioral events triggered by single components
(Silverstein 1984). Synergetically active compounds may
be inactive when o€ered individually.
Aliphatic hydrocarbons were most abundant in the
surface extracts of virgin gynes and breeding queens.
Cuticular hydrocarbons have been shown to be involved
in nest and nestmate recognition in termites (Howard
et al. 1982; Bagneres et al. 1990), ants (Bonavita-
Courgourdon et al. 1987; Soroker et al. 1995), wasps
(Lorenzi et al. 1997; Singer and Espelie 1997) and honey
bees (Getz and Page 1991; Page et al. 1991; Breed and
Stiller 1992), all highly eusocial insect groups. In the
primitively eusocial species L. (Evylaeus) malachurum we
104
found a similar function. The hydrocarbon pattern was
characteristic in females belonging to di€erent castes or
functional groups (Ayasse et al. 1991). Hydrocarbons
were also involved in the colony recognition cues used
by homing bees to identify their nest entrance (Ayasse
1990, 1991) and were used by kleptoparasitic bees to
recognize host nests (Sick et al. 1994). Until now
hydrocarbons could not be identi®ed as sex pheromone
components in bees (Dueld et al. 1984; Vander Meer
et al. 1998). In L. (Evylaeus) malachurum, n-alkanes and
n-alkenes attracted males to the odor source, and syn-
thetic mixtures of hydrocarbons released 80% of male
reactions compared with the complete volatile bouquet
on the cuticle. However, copulatory attempts were
released by other compounds like isopentenyl esters of
unsaturated fatty acids and unsaturated macrocyclic
lactones, which were identi®ed on the cuticle of attrac-
tive young gynes in small amounts only. Therefore, the
main role of hydrocarbons might be to provide infor-
mation on conspeci®c identity as well as on e.g., nest-
mates, individual age, and caste, as was shown in several
social insect species.
Although wax-type esters have also been identi®ed in
bumble bees (Ayasse et al. 1995; Hefetz et al. 1996),
honey bees (Katzav-Gozansky et al. 1997), and solitary
bees (Sick et al. 1994), almost no communicative func-
tion has been described. Hexadecyl oleate was the main
compound identi®ed on the cuticular surface of virgin
gynes and also in males (Ayasse 1991, 1994). Interest-
ingly, in ®eld experiments, a synthetic sample of this
compound was not attractive to males (M. Ayasse, un-
published data). When cuticular extracts of an attractive
gyne were o€ered simultaneously with hexadecyl oleate
in the same concentrations as found in males, it even
inhibited copulatory attempts (Ayasse 1994). Headspace
samples of mating bees showed this compound to be
released by the bees during copulation (Ayasse
1994).Therefore hexadecyl oleate seems to have a com-
municative function in signaling to conspeci®c males the
mating status of a female during copulation.
Macrocyclic lactones are abundant in the Dufour's
glands of halictine bees (Cane 1981; Johansson et al.
1982; Smith et al. 1985; Hefetz 1987, 1990; Hefetz and
Graur 1988). In L. (Evylaeus) malachurum, Ayasse et al.
(1993) showed that these compounds are not produced
at high rates throughout life. Instead, lactone produc-
tion is clearly correlated with the use of macrocyclic
lactones to line the walls of the brood cells and the nest
entrance tube. The age-speci®c patterns of volatile
compounds in queens revealed that macrocyclic lactones
were the main components only in breeding queens with
large ovaries and in workers (Ayasse et al. 1990a, 1993).
A study of con¯ict over nest ownership among
L. (Evylaeus) malachurum queens during the solitary
phase of the nest cycle demonstrated that lactones in
addition to lining the walls of brood cells can signal size
(Smith and Weller 1989). Whether lactones are used in
dominance interactions of nestmates is presently under
investigation (M. Ayasse, unpublished data).
Composition and modulation of the female sex
pheromone in L. (Evylaeus) malachurum:
how mating expenditures are economized
According to our experimental data, the female sex
pheromone in L. (Evylaeus) malachurum is a complex
mixture of chemical compounds signaling di€erent
messages over long-range, short-range, and touch-range
distances. The long-range message is probably the label
of a conspeci®c queen, di€ering from the worker odor
(Ayasse 1991; Ayasse et al. 1993). The short- and touch-
range message, which releases pouncing and copulation,
is evidently deposited on the abdominal cuticle and may
be produced by several glands. Isopentenyl esters are the
main constituents of this ®nal mating signal. Shortly
after copulation (3 h or even less), the inseminated
young gyne drastically reduces the emission of the
isopentenyl esters in particular; this modulation of
female sex pheromone composition enables a young
gyne to de®nitively inform the males about her receptive
status.
Mating strategies always evolved in directions which
increased ®tness (Emlen and Oring 1977). The speci®c
lek situation in L. (Evylaeus) malachurum requires an
e€ective economization of the mating tactics in both
sexes. Competition among the males should create se-
lection that proximately rewards males capable of pre-
cisely recognizing promising mates, and should
ultimately lead to a mating behavior where males spend
no time unsuccessfully chasing already mated queens.
Selective pressure on the female side ultimately operates
in the direction of a pheromonal gestalt which, except
during a rather short period of receptiveness, always and
perfectly repels the mate-searching males; the proximate
result is an undisturbed female after mating. In fact,
mated L. (Evylaeus) malachurum gynes are recognized
but not chased by the males. Therefore, the females do
not lose the time and energy they require to provide
themselves with pollen and nectar necessary to survive
the winter diapause, which lasts for up to 7 months
(Knerer 1992).
Acknowledgements The authors wish to thank Jan TengoÈ and
Hannes Paulus for valuable comments on the manuscript. This
work was supported by the Deutsche Forschungsgemeinschaft (En
89/11, Fr 507/8) and the FWF Austria (P09773-BIO).
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