Behav Ecol Sociobiol (2005) 58: 270–276
DOI 10.1007/s00265-005-0923-9
ORIGINAL ARTICLE
Roi Dor · Tamar Katzav-Gozansky · Abraham Hefetz
Dufour’s gland pheromone as a reliable fertility signal among
honeybee (Apis mellifera) workers
Received: 3 November 2004 / Revised: 17 January 2005 / Accepted: 25 January 2005 / Published online: 19 March 2005
C Springer-Verlag 2005
Abstract When a colony becomes queenless and without
the possibility of requeening, honeybee workers initiate
reproduction and lay male eggs about a week later. Assays in which two bees were confined in a small arena
revealed that they establish a reproductive dominance hierarchy, i.e., one worker demonstrates greater ovarian development than her paired bee. Reproductive dominance
is independent of relatedness, and can be established between full sisters, cousins, or random nestmates. A social
environment, however, is obligatory, as singly housed bees
fail to develop ovaries on the same time scale. Allowing
varying degrees of social interactions between the paired
bees revealed that olfaction of volatile bee compounds, as
well as tactile communication, seem to provide the necessary social environment. Ovarian development was accompanied by the production of queen-like Dufour’s gland
secretion in these workers. Especially notable was the increase in the queen-like esters. This increase was tightly
linked to ovarian development and not necessarily to the
dominance status of the bees in the pair. Thus, the occurrence of queen-like esters can serve as a reliable fertility
signal. Advertising ovarian status may recruit helper workers with less developed ovaries (and which are less likely to
successfully reproduce before colony breakdown) to assist
their nestmates and thereby gain inclusive fitness. Revealing the role of Dufour’s gland secretion as a fertility signal
adds another dimension to our understanding of how queen
pheromones operate. The mandibular-gland secretion is a
good predictor of dominance hierarchy, being correlated
with false-queen characteristics but not fertility, whereas
Dufour’s gland secretion is a good predictor of fertility but
not dominance hierarchy.
Communicated by R.F.A. Moritz
R. Dor () · T. Katzav-Gozansky · A. Hefetz
Department of Zoology, George S. Wise Faculty of Life
Sciences, Tel Aviv University,
Ramat Aviv,
69978 Tel Aviv, Israel
e-mail: [email protected]
Tel.: +972-3-6408766
Fax: +972-3-6406991
Keywords Dufour’s gland . Reproductive dominance .
Honeybee . Fertility signal
Introduction
Honeybees present an example of extreme reproductive
skew, where the queen is considered as the sole reproductive female in the hive. This is largely attributed
to the kin-selected “worker policing” that has evolved
to counteract reproductive attempts by selfish workers.
Since in honeybees the queen is multiply mated, selfish
workers’ reproduction under queenright (QR) conditions
reduces the inclusive fitness from male production of
less related workers, promoting policing of these eggs.
(Woyciechowski and Lomnicki 1987; Ratnieks 1988;
Ratnieks and Reeve 1992). Worker policing is manifested
through oophagy of worker eggs (Ratnieks and Visscher
1989; Ratnieks 1993; but see Pirk et al. 2004 for reservations regarding oophagy as a means of worker policing),
as well as aggressive behaviour towards reproductively developed workers (Sakagami 1958; Velthuis 1976; Visscher
and Dukas 1995). Consequently, under QR conditions,
only a few worker-laid eggs develop into males (Ratnieks
and Visscher 1989; Visscher 1989, 1996). This has
theoretically selected for workers exercising reproductive
self-restraint (Ratnieks 1988; Wenseleers et al. 2004).
However, when the colony has lost its queen and fails
to raise a replacement (hopeless-queenless situation, QL),
worker reproduction becomes the only way to gain fitness.
Soon after the colony becomes QL, several workers start to
develop ovaries and lay eggs (Page and Erickson 1988). Kin
selection theory predicts that under these conditions, workers will compete among themselves over male production.
This competition leads eventually to colony breakdown,
allowing only a narrow window for worker reproduction.
Workers may theoretically either compete for gaining
direct fitness by reproducing and inhibiting other workers,
or by helping nestmates to reproduce and thereby gain
inclusive fitness. Competition for reproductive dominance
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may be accompanied by aggressive behaviour among the
workers, and sometimes leads to the development of “pseudoqueens” (Sakagami 1954, 1958; Page and Erickson 1988;
Van der Blom 1991). However, it is not yet clear which factors determine the identity of the workers that successfully
reproduce under QL conditions. Differential reproductive
success of QL workers was shown to be related to patriline
differences, both with regard to the dynamics of ovarian
development and egg acceptance by other workers (Moritz
et al. 1996; Martin et al. 2004). Whether worker-brood
rearing is also kin-biased is not clear yet, but recent
findings that QL workers form kin-biased cliques with
respect to spatial distribution (Meixner and Moritz 2004)
lend credence to this hypothesis. Another still unsolved
question is that of how reproductive workers are recognized by their nestmates. We hypothesize that concomitant
with ovarian development, workers produce and emit
a fertility signal. Less physiologically apt workers that
detect the fertility signal consequently assist the advanced
reproductive workers to rear sons and gain at least some
inclusive fitness through that act.
Establishment of dominance hierarchies is a common
phenomenon in social Hymenoptera, and it can be mediated through behavioural interactions and/or pheromones
(Monnin et al. 1998; Bloch and Hefetz 1999; Cuvillier-Hot
et al. 2001; Sledge et al. 2001; Heinze et al. 2002).
Among the pheromone signals that are likely to mediate
worker-worker competition in honeybees, queen-like
pheromones are excellent candidates. At least two of
them, the queen mandibular pheromone (QMP) and the
tergal gland secretion, are implied in regulating ovary
development in workers (Butler 1959; Jay 1968; Velthuis
1970; Wossler and Crewe 1999; Hoover et al. 2003).
Dufour’s gland is another source of queen-like pheromone
that, like the two former pheromones, is highly attractive
to workers, which display retinue behaviour around the
secretion (Katzav-Gozansky et al. 2001, 2002). In addition,
the honeybee queen pheromones are not produced by a
“fixed caste-specific pathway”, but show great plasticity
in workers. When the queen is lost, some of the workers
start to produce the queen-like substances in both the
mandibular (Crewe and Velthuis 1980; Plettner et al. 1993)
and Dufour’s (Katzav-Gozansky et al. 1997) glands. If the
production of a queen-like compound in workers signals
fertility, these two phenomena should be invariably linked.
While this was demonstrated for Dufour’s gland secretion
(Katzav-Gozansky et al. 1997, 2004), it is still controversial for the mandibular gland secretion. Some reports show
a positive correlation (Crewe and Velthuis 1980), whereas
others demonstrate that workers with activated ovaries still
produce and emit a worker-like secretion (Hemmling et al.
1979; Hepburn et al. 1988). A later report has indicated that
among workers that have developed ovaries, only those
classified as pseudoqueens had queen-like mandibular
gland secretion (Plettner et al. 1993). Recent studies with
Apis mellifera capensis have shown that QL workers
housed in pairs indeed established dominant-subordinate
relationships with respect to the production of 9-oxo2-decenoic acid, a queen mandibular gland component
(Moritz et al. 2000, 2004). Since QMP is reported to inhibit
ovarian development, it was assumed that pheromonal
dominance also reflects reproductive dominance; however,
the ovarian state of these workers was not reported.
In order to assess the relationship between dominance
hierarchy, ovarian development and Dufour’s pheromone
production, we used Italian bees in paired-bees contests. Although this experimental approach is highly
reductive, it enabled manipulation, as well as precise
measurement of ovarian development and pheromone
quantities that would be hard to obtain in full-size
hives.
Methods
Bees
All experiments were conducted using A. m. ligustica
workers obtained from commercial hives kept in the
Tzriffin apiary, Israel and in the I. Meier Segals Garden
for Zoological Research at Tel Aviv University, during
2002 and 2003. All hives had naturally mated queens,
unless stated otherwise. The experiments were conducted
using callow bees less than 12 h old that had emerged
from sealed brood combs in an incubator. The experiments
were carried out at a constant temperature of 33◦ C and
60–70% humidity in darkness (except during behavioural
observations that were done under normal light).
Experimental set-up
Reproductive development among random pairs
and single bees
Reproductive dominance among workers was investigated
using callow workers that were maintained in pairs for
10 days. Preliminary results had shown this time period
to be sufficient to allow full ovarian development under
these conditions. Pairs were selected at random (n=25
pairs, termed “random pairs”, and n=44 single bees from
3 hives for experiment 1; and n=89 pairs, termed “unrestricted pairs”, from 3 hives for experiment 2). The
bees were kept in a circular arena (9 cm petri dish)
lined with filter paper, and were supplied with pollen
and candy (sugar mixed with honey) ad libitum. To assess the rate of ovarian development independent of social
interactions, bees were also kept singly using the same
set-up.
Relatedness effect on reproductive dominance
In order to test the effect of the bees’ patriline (same or
different patriline) on reproductive dominance, this experiment was repeated using bees descendent from three singledrone-inseminated queens. The queens utilized were sisters and the inseminating drones were brothers, but from a
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different hive. Pairs of workers were composed either of full
sisters or cousins. Singly isolated bees were concomitantly
established as described above.
then held for 10 min. Compound quantification was done
by peak integration in comparison to the internal standard.
Statistical analysis
Social interaction effect on reproductive dominance
To further investigate how social interactions affect worker
ovarian development, workers were divided into different experimental groups: (1) singly isolated bees (“single
bees”); (2) a pair of bees (“unrestricted pairs”); (3) two bees
separated by a single 1-mm mesh screen, dividing the arena
into two halves, allowing antennal contact and trophallaxis (personal observations, data not presented) through
the mesh (“SM bees”); (4) two bees separated by a single
mesh as in (3), but allowed to interact for 1 h daily by
removing the screen (“SM bees+encounter”); and (5) two
bees separated by a double screen, 1 cm apart, which prevented all direct contacts but allowed volatile substances to
pass through (“DM bees”).
Detailed behavioural observations were conducted with
the two groups in which the bees were able to interact directly (groups 2 and 4 above). Observations were performed
daily for 5 min per pair. For the “SM bees+encounter”
group, observations started after screen removal, whereas
for the “pairs” group they were scheduled randomly.
For each session, the interactions between the bees were
recorded every 10 s (30 observations per session), including
head and body antennation, body–body contact, trophallaxis, grooming, biting and head bumping. Interaction levels were calculated as mean number of interactions for
each observation-group per day. Preliminary observations
did not reveal a correlation between the role of the bees
(donors or receivers) during trophallaxis and reproductive
dominance, although such correlation has been found in a
previous study (Korst and Velthuis 1982).
Statistical analyses were performed using Statistica for
Windows, version 6.0, Statsoft. Ovarian development in
paired bees and trophallaxis levels between the ages of
5 and 6 days in control and mesh-separated pairs were
compared using Wilcoxon matched pairs test. This test
was also used to compare overall trophallaxis levels between control and mesh-separated bees. Mann-Whitney Utest was used to compare ovarian development between
the “single bees” and bees from “unrestricted pairs” that
exhibited reduced ovary development (defined as subordinate bees). Nonparametric Kruskal-Wallis ANOVA and
Tukey-type post-hoc tests (Zar 1996, Dunn equation, 31,
p 227), were used to compare ovarian development of full
sisters, cousins and random pairs with developed ovaries
(defined as dominant bees), among dominant bees at the
different levels of isolation, as well as “single bees”, and
to compare hive origin of dominant bees. T-test for independent samples was used to compare the interaction
levels of control and mesh-separated bees and to compare
the amount of secretion between bees with developed or
undeveloped ovaries. Two-way ANOVA and Bonferroni
post-hoc tests were used to evaluate the effect of both absolute ovary developmental stage and dominance versus
subordinate ovary developmental stage on ester quantity.
Kendall tau correlations were used to correlate ovary developmental stage and esters to hydrocarbon ratios, and to
correlate trophallaxis levels with the bee’s age. Statistical
significance was accepted at α=0.05. Data are presented as
means±SE.
Results
Ovarian development
After 10 days of experiment, the bees were frozen and
kept at −20◦ C until dissection. They were dissected under a binocular microscope in double distilled water, and
ovarian development was scored on a 1–3 scale: stage
1=undeveloped ovaries, stage 2=small rounded eggs, stage
3=large oval eggs (Velthuis 1970).
Chemical analyses
Chemical analyses of Dufour’s gland secretion were done
using the bees from the “unrestricted pairs” (n=89 pairs).
Dufour’s gland was extracted in 50 µl dichloromethane
containing 200 ng eicosane as internal standard. Chemical
composition of Dufour’s gland secretion was verified using
GC/MS according to Katzav-Gozansky et al. (1997). Quantitative analyses were conducted by GC (Varian CP 3800)
using a DB-1 fused silica column that was temperature
programmed from 150 to 300◦ C at a rate of 5◦ C/min and
Bees housed in pairs established dominant-subordinate hierarchies with regard to ovarian development. Figure 1
gives the distribution of ovarian development in the paired
workers, classified as either dominant or subordinate, from
experiments 1 and 2 combined (n=114 pairs) after 10 days.
Of all bee pairs, 39 were of bees that had equal ovarian development and their status, dominant or subordinate, was
assigned randomly. These included 8, 7 and 24 pairs of
bees that had ovaries at stages 3, 2 and 1, respectively.
Taking all bees into account, Fig. 1 shows that the distribution of dominant bees was mainly at ovary stages 2
and 3, whereas the distribution of the subordinate bees was
mainly at ovary stage 1. In experiment 1, in most cases (18
out of 25 pairs), 1 of the paired bees had more developed
ovaries (dominant) than her partner (subordinate) (Fig. 2,
random pairs; average stage 2.36±0.16 for the dominant
vs 1.48±0.15 for the subordinate bees; Wilcoxon matched
pairs test T=0.0, n=25, P<0.001). In five pairs, both bees
had undeveloped ovaries (stage 1), and in two pairs both
had stage 3 ovaries. In experiment 2, in 57 out of the 89
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Fig. 1 Distribution of ovarian development in paired workers kept
in petri dishes for 10 days. Ovarian development was classified as:
1 undeveloped, 2 medium developed, 3 fully developed. For each pair,
the bee with greater ovarian development was classified as dominant
and the bee with lesser ovarian development as subordinate. Bees
that had equal development (8, 7, and 24 pairs with ovaries at stages
3, 2, and 1, respectively) were randomly classified as either dominant
or subordinate. Data are pooled from experiments 1 and 2 (n=114
pairs: exp. 1 n=25 pairs termed “random pairs” and exp. 2 n=89
pairs termed “unrestricted pairs”)
Fig. 2 The effect of patriline origin on reproductive dominance
establishment in paired bees. Bees were kept in pairs or singly for
10 days. Bees termed “random pairs” were obtained from the same
hive, patriline unknown. Bees obtained from the same hive headed
by a single-drone-inseminated queen (SDI hive) are full sisters. Bees
taken from different SDI hives are cousins (queens of each hive were
sisters and the inseminating drones were brothers). The numbers on
top of the bars represent the number of groups (pairs/single bees).
Bees in pairs marked with an asterisk established a significant dominance hierarchy with respect to ovarian development (*Wilcoxon
matched pairs test P<0.001)
pairs, the bees established reproductive dominance (Fig. 3,
unrestricted pairs; average 2.16±0.08 for the dominant vs
1.31±0.06 for the subordinate bees; Wilcoxon matched
pairs test, T=0.0, n=89, P<0.0001). In 6, 7 and 19 pairs,
both bees had equal ovary development at stages 3, 2, and
1, respectively.
Since the bees for experiment 1 were taken from twostorey-hives with naturally mated queens, the probability
that the bees in a pair belonged to different patrilines was
high (Page and Metcalf 1982). It can be argued therefore
that dominance establishment may have reflected the coupling of a bee from a patriline that showed predisposition
to reproductive dominance, with a bee from a patriline
that showed less predisposition to such. To test this
Fig. 3 Ovarian development in paired bees experiencing various
degrees of social isolation as well as singly isolated bees (for abbreviations see Methods). The numbers on top of the bars represent the
number of groups (pairs/single bees). Bees in pairs marked with an
asterisk established a significant dominance hierarchy with respect
to ovarian development (*Wilcoxon matched pairs test P<0.001).
Different letters denote significant differences between the dominant
bees of each group and the single bees (Kruskal-Wallis ANOVA
H4,376 =190.2, P<0.001 followed by Tukey-type post-hoc test at
P<0.05)
hypothesis, we repeated the experiment with descendants
of three single-drone-inseminated queens (hives A–C) and
pairing either full sisters or cousins. Figure 2 shows that
similar reproductive dominance was obtained between full
sisters, as well as between bees from different hives, i.e.,
cousin bees (super-sisters: average stage 2.04±0.10 for the
dominant vs 1.38±0.08 for the subordinate bee; cousinbees: 1.93±0.10 vs 1.38±0.08, respectively, Wilcoxon
matched pairs test, same hive: T=0.0, n=56, P<0.001,
different hives: T=0.0, n=58, P<0.001). There was no
significant difference in ovarian development between the
dominant bees of the full sisters, cousins and random pairs
(Kruskal-Wallis H2,139 =5.42, P>0.05). However, not all
three hives showed equal worker reproduction potential.
The dominant bees from hives A and B had significantly
greater ovary development than the dominant bees
from hive C (average ovary stage; hive A: 2.15±0.11,
hive B: 2.08±0.11, hive C: 1.52±0.13, Kruskal-Wallis
H2,56 =13.2, P<0.01; post-hoc test P<0.01 for A vs C
and P<0.05 for B vs C), but there was no difference
between the dominant bees of hives A and B (post-hoc test,
P>0.05). Nonetheless, bees from hive C did not become
automatically subordinate when paired with bees from
either hives A or B. When paired with A or B, C-bees became dominant in 5% and 10.5% of the cases, respectively,
slightly lower compared to the 35% of cases in which
B-bees paired with A-bees became dominant. Dominance
hierarchy was also established when full sisters from hive C
were paired (Wilcoxon matched pairs test, P<0.01, n=19).
In order to determine the degree of ovarian development
in workers independent of social interactions, singly housed
bees were also monitored. However, these showed almost
no ovary development even after 10 days (average stage
1.07±0.03, n=70, Fig. 2).
The results obtained with singly housed bees suggested
that social interactions were important for reproductive development in workers. To test this hypothesis, we repeated
the pairing experiments, but introduced graded levels of
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social isolation between the paired bees (Fig. 3). Ovarian development in dominant bees belonging to the
“unrestricted pairs” group was significantly higher than
in other treatments (except for the “SM bees” which
although lower, was not statistically significant) and in
the single bees (Kruskal-Wallis H4,376 =190.2, P<0.001
followed by post-hoc test at P<0.05). The dominant
bees from all tested groups had significantly greater
ovarian development in comparison to the “single bees”
(see statistics results above). Nevertheless, dominance
hierarchy was established also in the socially restricted
groups (Wilcoxon matched pairs test, T=0.0 “unrestricted pairs”: n=89, P<0.0001, “SM bees”: n=60,
P<0.0001, “SM+encounter bees”: n=42, P<0.001,
“DM bees”: n=65, P<0.0001). Ovarian development
among the subordinate bees was low with high variability
(Kruskal-Wallis H4,376 =13.4, P<0.05), but post-hoc
tests revealed that differences between groups were not
significant (P>0.05).
Behavioural observations revealed that the frequency
of interactions was higher among bees from the
SM+encounter pairs than among bees from the unrestricted pairs (12.5±1.1 average number of interactions
and 6.2±0.8, respectively; t-test for independent samples: T=17.2, df=9, P<0.0001). Trophallaxis was the
most remarkable behaviour observed. For both groups,
its proportion (out of total interactions) increased during
the first 5 days (Kendall tau correlations, “unrestricted
pair”: Kendall tau=0.29, P<0.001, “SM+encounter bees”:
Kendall tau=0.36, P<0.001), then dropped to a lower level
on day 6 (Wilcoxon matched pairs test: “unrestricted pair”:
T=25, n=33, P<0.05, “SM+encounter”: T=146, n=46,
P<0.05). This level was retained for the remainder of the
experiment. However, the level of trophallaxis interactions
was higher among the “SM+encounter bees” (Wilcoxon
matched pairs test T=0.0, n=10, P<0.01).
Chemical analysis of Dufour’s gland secretion of the
“unrestricted pairs” group (Fig. 4) showed a gradual
increase in the total secretory amounts concomitant with
an increase in ovarian development. Bees with developed
Fig. 4 Total amounts of Dufour’s gland secretion and esters to hydrocarbons ratios according to ovarian development in bees housed as
“unrestricted pairs”. All bees were considered irrespective of whether
they were dominant or subordinate within the pairs. The numbers
on top of the bars represent the number of bees. Compound quantification was performed by gas chromatography using extracts of
individually dissected glands. For details of analyses see Methods
ovaries (stage 3) had a significantly higher amount of secretion compared to workers with undeveloped ovaries (stages
1+2) (711±55 vs 259.7±64 ng/gland, mean±SE, respectively; t-test for independent samples: T=3.05, df=176,
P<0.01). There was a positive correlation between ovary
developmental stage and the esters to hydrocarbons ratios
(Kendall tau correlations, Kendall tau=0.32, P<0.00001).
However, glandular chemistry (i.e., ester amounts) was not
correlated with the bee’s social status, i.e., dominant vs
subordinate (two-way ANOVA, F=0.01, df=1, P=0.92),
but only with the absolute stage of ovary development
(two-way ANOVA, F=3.88, df=3, P<0.01).
Discussion
When a honeybee colony becomes hopelessly QL, worker
reproduction is initiated, resulting in male production. This
is accompanied by worker–worker competition that eventually leads to the social breakdown of the colony, with only a
narrow opportunity window for successful rearing of males
(Sakagami 1954, 1958; Page and Erickson 1988; Van der
Blom 1991). Worker–worker competition generates several
predictions as to how workers behave to maximize fitness.
Workers that are physiologically more primed for rapid
ovarian development will concomitantly develop means to
inhibit lesser physiologically primed nestmates from reproducing. The latter, which have little chance of rearing
progeny before colony breakdown, will at least gain inclusive fitness by becoming helpers. Effective inhibition thus
increases the hive’s reproductive efficiency and permits
successful rearing of a greater number of males. Such inhibition should therefore be favourably selected, as should the
evolution of a reliable signal indicating which of the workers are ahead in their ovarian development. In the present
study, we attempted to uncover whether reproductive dominance exists among QL workers of A. m. ligustica, and if
so, to evaluate its possible pheromonal correlate, namely,
Dufour’s gland secretion.
Reproductive dominance, as expressed by ovarian development, was induced between paired workers after 10 days.
Although we did not wait for actual egg-laying (honeybees
do not lay eggs in the absence of a comb), it is very likely
that the dominant worker would have won the competition and laid eggs before her partner. The few pairs that
did not develop dominance hierarchy were of two types. In
most pairs neither bee had developed ovaries. This may reflect the pairing of less physiologically apt bees. In contrast,
when both bees in a pair had equally developed ovaries, this
may have been due to pairing particularly physiologically
primed bees that resisted mutual inhibition. The fact that
full sisters also established a dominance hierarchy, which
was not significantly different from that in random pairs,
excludes the possibility that pairing bees from different
patrilines caused the dominance effect. We attribute dominance establishment to true competition over reproduction
among workers. However, the occurrence of a patriline
that showed lesser ovarian development suggests that the
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patriline effect may still bias worker reproduction in hives
with multiply mated queens (Robinson et al. 1990; Page
and Robinson 1994; Martin et al. 2004).
Another striking phenomenon in worker reproduction is
the need for a social environment. Bees housed singly for
10 days failed to developed ovaries. This cannot be attributed to lack of resources since the bees received pollen
and candy ad libitum and were observed to feed normally
and not differently from the bees in the “unrestricted pairs”.
Since bees that were separated by a DM could not have
had any physical contact with each other, but nevertheless at least one of the paired bees still developed ovaries,
we suggest that volatile odours provide the social environment. In addition, the fact that socially restricted bees had
less-developed ovaries compared to unrestricted bees suggests that tactile communication is important too. This was
also substantiated by the behavioural observation. Both the
“unrestricted pairs” and the “SM+encounter” bees were
engaged in social interactions, in particular trophallaxis;
albeit in the latter these were more frequent. Since both
bees had unlimited access to food, we suggest that trophallaxis may have served as a communicative channel between the two bees, confirming earlier studies (Korst and
Velthuis 1982). However, unlike the previous study, which
implied that bees with developed ovaries tend to receive
more trophallaxis than they give, in our preliminary observations no such a correlation was found. The need for
social interaction for triggering queen-like characteristics
in workers was also described in A. m. capensis, in which
bees in pairs produced large amounts of 9-ODA, whereas
singly isolated bees failed to do so (Moritz et al. 2000).
However, since in those studies ovarian development was
not explicitly studied, we can only infer that in A. m. capensis too, “single bees” fail to develop ovaries, since workers
that possess 9-ODA also have developed ovaries (Crewe
and Velthuis 1980; Moritz and Hillsheim 1985).
Our experiments revealed that Dufour’s gland secretion
is correlated with ovarian development. Workers with developed ovaries had both higher amounts of secretion and
higher esters to hydrocarbons ratios, indicating that the
increase in secretory volume can be attributed mostly to
boosting of queen-like ester production in these workers.
These results are consistent with data obtained from minihives containing several thousand bees (Katzav-Gozansky
et al. 2004). However, in the latter experimental set-up, the
possible roles of the queen-like esters as a fertility and/or
dominance hierarchy signal may have been confounded.
By confronting only two bees and establishing unequivocally the reproductively dominant individual, we were able
to determine that Dufour’s esters seem to act solely as
a fertility signal. Ester quantity was positively correlated
to the degree of ovarian development, but uncorrelated to
the hierarchical status of the bees. This is in contrast to
the mandibular gland secretion, which is uncorrelated with
worker ovarian development, i.e., egg-laying workers may
have low amounts, but this is positively correlated with
dominance hierarchy; false queens possess high amounts
(Plettner et al. 1993). This suggests that the queen honeybee uses two different pheromones, one for denoting fer-
tility and the second for establishing dominance over her
workers. These two effects may not necessarily be mutually exclusive but, however, may be additive or even act in
synergy.
The use of a queen-like signal to denote ovarian development was also recently reported in the ant Pachycondyla
inversa (Heinze et al. 2002; D’ettorre et al. 2004). Although
it is difficult to generalize from these two systems, queen
pheromone plasticity in workers is worth noting. The adaptive significance of such pheromone plasticity is evident.
By signalling their reproductive status, competing workers
may gain the cooperation of less fecund workers and fully
exploit the narrow reproduction window between queen
loss and colony loss. Helpers, however, by responding to
the signal gain inclusive fitness. Helper bees are not necessarily kin, as might have been predicted from kin-selection
theory. Our single-drone-insemination experiment did not
reveal kin preference, but did show that certain patrilines
may produce less physiologically apt workers, at least with
respect to reproduction. This result confirms an earlier finding of differential reproductive success of workers from
different patrilines (Martin et al. 2004), and further suggests that the cause is physiological aptitude rather than
behavioural dominance. However, we cannot exclude the
possibility that in larger groups kin-based helping groups
occur (Meixner and Moritz 2004).
Using a reductionist approach, e.g., confronting only two
bees, enabled us to clearly demonstrate that in the highly
socially advanced honeybees too, reproductive dominance
among workers is maintained. We also show that Dufour’s
gland plasticity may act as a fertility signal. Although these
experiments did not allow us to determine whether the
changes in Dufour’s gland signalling precede ovarian development or are an outcome of this development, based
on these findings, we can postulate that queens too utilize Dufour’s gland secretion to denote fertility. If the
amount of Dufour’s secretion is correlated with the degree of queen fertility as this and previous studies suggest
(Katzav-Gozansky et al. 1997, 2004), it may enable workers to detect reproductively declining queens and supersede
them. Moreover, covariance between Dufour’s gland secretion and ovarian development may assist workers to locate
other workers that attempt to reproduce under QR condition, and punish them accordingly (Heinze et al. 2002 for
P. inversa; Visscher and Dukas 1995 for A. mellifera).
Finally, the finding that two distinct pheromones may
denote separately dominance (i.e., the mandibular gland
secretion) and fertility (i.e., Dufour’s gland secretion)
may add building blocks to our understanding of queen
pheromone evolution. It is clear that, in the society, the
queen is portrayed by multiple pheromones, but whether
these evolved in concert or sequentially is still obscure. By
using simple, albeit reductionist, systems, the proximate
as well as ultimate factors affecting queen pheromones
may emerge.
Acknowledgements This research was supported by the Israel Science Foundation founded by the Israel Academy of Sciences and
Humanities. The authors wish to thank two anonymous reviewers for
276
their helpful comments. We thank Tovit Simon for her technical help,
Armin Ionescu for his statistical assistance, Josef Kamer and Haim
Efrat from Tzrifin Apiary for assistance in establishing experimental
hives, and N. Paz for editorial assistance.
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