ANIMAL BEHAVIOUR, 2005, 69, 427–435
doi:10.1016/j.anbehav.2004.03.017
Worker nutrition and division of labour in honeybees
A MY L. TOTH * & G ENE E. ROB IN SON *†
*Program in Ecology and Evolutionary Biology, University of Illinois at Urbana-Champaign
yDepartment of Entomology and Neuroscience Program, University of Illinois at Urbana-Champaign
(Received 4 September 2003; initial acceptance 4 December 2003;
final acceptance 23 March 2004; published online 16 December 2004; MS. number: A9692)
We determined whether there is an association between nutritional state (as indicated by stored abdominal
lipid amounts) and division of labour in the honeybee, Apis mellifera. We found that foragers (typically
older bees) had lower lipid amounts than did nurses (typically young bees). Results from experimental
colonies that contained nurses and foragers of the same age showed that the lipid decline in foragers was
not attributable to age. Analysis of bees with different amounts of foraging experience revealed little effect
of the act of foraging on lipid stores. Lipid levels were low even on the first day of foraging, suggesting that
the decline in stored lipid precedes the onset of foraging. We also found that bees that revert from foraging
to nursing did not regain their lipid stores, indicating that high lipid stores are not required to sustain
brood care behaviour. This demonstration of a robust association between reduced lipid stores and the
transition to foraging suggests that worker nutritional state may be involved in the regulation of division
of labour in honeybee colonies.
Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Colonies of advanced social insects show two forms of
division of labour: a division of labour between queens
and workers for reproduction and a division of labour
among workers for tasks related to colony growth and
development (Robinson 1992). In the social Hymenoptera, reproductive division of labour between morphologically distinct queens and workers involves differences in
nutrition during larval development (Wheeler 1986;
Hartfelder & Engels 1998). Division of labour between
morphologically distinct worker castes in ants also relates
to differences in larval nutrition (Wheeler & Nijhout 1983).
Nutritional differences have been shown to be correlated
with behavioural division of labour among morphologically identical workers (Blanchard et al. 2000), but the role of
nutrition in these systems is less understood than for
systems that involve morphological differentiation.
Many social insect species show age-related division of
labour, whereby each worker progresses through a set of
behavioural changes over her life, usually involving
a transition from nest work to foraging (Wilson 1971).
This involves not only drastic changes in behaviour, but
sometimes also physiological changes such as the development of glands for brood feeding (Winston 1987)
Correspondence: A. L. Toth, Department of Entomology, 505 S.
Goodwin Avenue, 320 Morrill Hall, Urbana, IL 61801, U.S.A. (email:
amytoth@life.uiuc.edu).
0003–3472/04/$30.00/0
and higher metabolism in foragers (Martin & Lieb 1979;
Harrison 1986). Although patterns of temporal polyethism are robust, worker behavioural development in several
species is flexible and can be accelerated, delayed, or
reversed in response to colony needs (Robinson 1992).
Since age can be uncoupled from behaviour, other factors
take precedence in the control of worker behavioural
development. Studies with honeybees have implicated
social, endocrine, neurochemical and genetic factors
(Robinson 2002).
In several species of social insects, nutritional differences among workers appear to be associated with task
performance. Porter & Jorgensen (1981) reported that
Pogonomyrmex owyheei ant foragers have substantially less
body weight than callow workers, and these authors
further suggested that depletion of body energy reserves
may stimulate foraging. Blanchard et al. (2000) found
a negative correlation between amounts of energy reserves
and foraging tendency in Leptothorax albipennis ants. The
pattern of ‘lean forager–corpulent nest worker’ appears to
be common in the social Hymenoptera, as it has been
observed in at least eight species of ants and two
wasps (see Tschinkel 1987, 1998; Blanchard et al. 2000;
Markiewicz & O’Donnell 2001). Based on their findings
and a review of other evidence from ants, wasps and bees,
Blanchard et al. (2000) proposed that differences in the
nutritional status of workers could be involved in the
regulation of division of labour. Supporting this idea,
427
Ó 2004 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
428
ANIMAL BEHAVIOUR, 69, 2
honeybees in starved colonies initiate foraging at significantly younger ages than well-fed bees (Schulz et al.
1998), suggesting that a change in worker nutritional state
could result in a behavioural change. However, the extent
to which differences in worker nutritional state influence
regulation of age-related division of labour is unknown.
Because nutrition can interact with numerous other
physiological processes, a better understanding of possible
nutritional factors is essential to the study of mechanisms
that affect division of labour.
The objective of this study was to determine whether
there is an association between nutritional state and
division of labour in the honeybee, Apis mellifera. We
focused on two distinct honeybee behavioural states,
brood care (nursing) and foraging, which represent easily
identifiable tasks performed early and late in behavioural
development, respectively. Nursing is typically performed
during the first 2 weeks of adult life, whereas foraging is
performed from about 2–3 weeks of age and proceeds for
the remainder of the typical 5–7-week life span (Winston
1987). As an indicator of nutritional state we measured
abdominal lipid, since energy stores in adult social
Hymenoptera are found mainly as lipid in the fat body
(Ricks & Vinson 1972). In experiment 1 we determined
whether honeybees show the lean forager–corpulent nest
worker pattern observed in other social insects (Blanchard
et al. 2000). In experiment 2 we examined whether
differences in lipid stores are associated with worker age
or behaviour. In experiment 3 we determined whether
abdominal lipid is affected by foraging, which is an
energetically demanding task. In experiment 4 we investigated whether bees that shift back from foraging to
nursing experience a corresponding change in lipid levels.
METHODS
Bees
Honeybees of a mix of European races were maintained
at the University of Illinois Bee Research Facility in
Urbana, Illinois, U.S.A. Experiments 1–3 were performed
between June and August 2001. Experiment 4 was performed between June and July 2002. We collected 1-dayold bees (experiments 1–3) by removing frames of comb
containing capped brood from source colonies with
multiply mated queens. We placed frames in a 33 C
incubator overnight and collected bees that had emerged
over a period of approximately 24 h for experiments. We
marked 1-day-old bees for later identification by painting
a small spot of Testor’s enamel paint on the thorax. All
bees collected for lipid analysis were killed by freezing in
a 80 C freezer and kept frozen at 80 C until assayed.
Lipid Assay
For each bee, we removed the entire digestive tract,
along with the sting apparatus and any wax scales
observed on the outside of the bee. Each abdomen was
freeze-dried at 300 mTorr (4 Pa) for 62 min, placed in 5 ml
of 2:1 chloroform:methanol (Folch extraction method,
Perkins 1975), homogenized using Kontes Tissue Grinders, and allowed to extract overnight. The extract was
filtered with glass wool and evaporated in a Savant Speed
Vac SC110 to a constant volume of 2 ml. We used a 100-ml
subsample of each extract for the lipid assay (developed
from Amenta 1964). We dried each sample completely,
then added 1 ml of reagent (0.25% potassium dichromate
in 80% sulphuric acid) and heated all samples for 20 min
in a bath of boiling water. We then diluted 100 ml of the
reaction product with 4 ml of distilled water and measured
absorbance of the sample using a Molecular Devices
SpectraMax 190 multiwell spectrophotometer at 350 nm.
We converted absorbance measurements to milligrams of
lipid using a cholesterol standard with each assay run.
Histological Examination of Fat Bodies
We examined fat bodies from individual bees histologically. We removed the abdomen, dissected out the digestive tract and sting, and cut a small square of cuticle
with adhered fat body tissue (visible with a dissecting
scope) from the middle of the second tergite of the gaster.
The fat body tissue was smeared on a slide, fixed with
formaldehyde, and stained with Oil Red O. We examined
the tissue under a compound microscope at 400! and
photographed it using an Olympus C3030 digital camera
at F 2.8, 1/30 s. Lipid staining was quantified using Adobe
Photoshop by selecting red pixels, which were counted
with the Histogram tool.
Experiment 1: do abdominal lipid stores
of nurses and foragers differ?
To compare lipid levels between nurse and forager bees,
we paint-marked 1-day-old bees and introduced them into
a typical field colony. Assessments of nursing and foraging
behaviour were made as described in Huang et al. (1994).
When marked bees were 7 days old, we collected a subset
of the bees observed performing nursing behaviour (bees
with their heads in cells containing larvae). When they
were 21 days old, we collected another set of bees observed
performing foraging behaviour (returning to the hive
carrying pollen or with abdomens distended with nectar).
Two trials were performed. In trial 1 only, fat bodies from
nurses (N Z 10) and foragers (N Z 10) were stained for
histology as described above.
Experiment 2: are abdominal lipid stores
associated with age or task performance?
To examine whether abdominal lipid stores are related
to age or task performance, we measured lipid stores of
nurses and foragers in single-cohort colonies at 7 and 21
days of age. We used single-cohort colonies because
a division of labour is established within a few days,
producing bees of the same age performing different tasks
(Robinson et al. 1989). To construct each colony, we
placed 1100–1200 1-day-old bees into a small hive box
with one empty frame of comb, one frame with an excess
of honey and pollen, and a naturally mated queen. When
bees were 7 days old, we collected precocious foragers and
TOTH & ROBINSON: NUTRITION AND DIVISION OF LABOUR
normal-age nurses. When bees were 21 days old, we
collected normal-age foragers and overage nurses (only
in trials 1 and 2). Three trials were performed.
Experiment 3: do abdominal lipid stores
vary with foraging experience?
We examined lipid levels in bees with different amounts
of flight experience. To control flight experience, we glued
plastic tags to the thoraces of bees and attached a screen to
the hive entrance to prevent tagged bees from leaving the
hive (Withers et al. 1995). Three treatment groups were
analysed: bees with unrestricted flight, partially restricted
flight, and totally restricted flight. Unrestricted bees were
not tagged but were paint-marked for later identification.
Partially restricted bees bore plastic tags that increased
their height by approximately 1.5 mm. Totally restricted
bees bore plastic tags that increased their height by
approximately 3 mm. One-day-old bees were collected
and randomly chosen to be in one of the three groups.
All three groups of bees were placed into a single Langstroth hive box containing an already established colony
with a typical age demography and approximately 10 000
bees. To restrict the flight of tagged bees, we used
a modified hive entrance with two screens, the first with
holes slightly larger than those in the second (Fig. 1).
Unrestricted bees passed through both screens and had
full flight experience. Partially restricted bees could pass
through the first screen, but not the second. The second
screen was removed every afternoon at 1300 hours, giving
partially restricted bees approximately a half day for
foraging. Totally restricted bees could not pass through
the first screen, and thus had no flight experience. We
collected bees from these three groups on the first,
seventh and 14th day following the onset of foraging by
unrestricted (control) and partially restricted bees. Marked
7-day-old nurse bees were also collected to provide
a baseline comparison. To validate the effects of tagging
on flight restriction, bees in the three flight experience
Unrestricted
Partially
restricted
groups were visually assessed for the presence or absence
of noticeable wing wear. Wing wear was defined as tears in
or small pieces missing from the wing margin. Honeybee
wings become tattered during flight, and wing wear can be
used to assess the extent of flight experience (Breed et al.
1990). One trial of this experiment was performed.
To assure there were no residual effects of carrying tags
on lipid levels, we performed a ‘control’ experiment in
which tagged bees were not restricted by screened entrances. Large plastic tags (3 mm high) were glued to the
backs of 1-day-old bees, and these bees were allowed
unrestricted flight by placing them in a pre-established
colony of approximately 10 000 bees with a normal hive
entrance. Tagged and untagged paint-marked bees were
collected for lipid analyses on the first, seventh and 14th
day following the onset of foraging.
Experiment 4: is behavioural reversion associated
with an increase in abdominal lipid?
To test whether foragers that revert to nursing behaviour experience an increase in abdominal lipid stores, we
set up ‘reversion colonies’ (Robinson et al. 1992). In
reversion colonies, a colony composed entirely of foragers
establishes a division of labour; some bees continue to
forage while others switch back to nursing behaviour
(reverted nurses). Reversion colonies were made as follows. We collected 2000 foragers from a typical field
colony (source colony). These bees were placed in a small
hive box with an empty frame, a frame with open brood
from the source colony, and the queen from the
source colony (as in Bloch & Robinson 2001). The new
reversion colony was moved several kilometres from the
source colony to prevent bees from returning to the
original colony. The colony entrance was left closed for
1 day (day 1 of the experiment) to allow bees to adjust to
the new colony environment. On days 2–4, we opened
the entrance and paint-marked all bees on the thorax that
we observed performing nursing behaviour. On day 5, we
Totally
restricted
1
2
Figure 1. Design of experiment 3. Honeybee flight from the hive was either unrestricted (untagged bees), partially restricted (bees with 1.5mm tags) or totally restricted (bees with 3-mm tags) using two screens (1 and 2) that modified the size of the entrance. Unrestricted bees could
pass through both screens, and had full flight experience. Partially restricted bees could pass through screen 1, but not screen 2. Screen 2 was
removed each afternoon, allowing partially restricted bees half of the day for foraging. Totally restricted bees could not pass through screen 1,
and thus had no flight experience.
429
ANIMAL BEHAVIOUR, 69, 2
collected reverted nurses and foragers for lipid analysis.
We identified reverted nurses as previously paint-marked
bees performing nursing behaviour. Two trials of this
experiment were performed.
Statistical Analyses
Statistical analyses were performed using SAS Software
(SAS Institute 2000). Lipid level measurements were
natural-log transformed to normalize the data. Extreme
outliers were excluded from analyses, and were identified
as those values with studentized residuals greater than 2.6.
In experiments with multiple trials (experiments 1, 2 and
4), we examined results from each trial separately and in
experiment-wide analyses. We used unpaired t tests (PROC
GLM) to analyse the lipid assay and staining data for
experiment 1, the trial 3 data for experiment 2, and all
data for experiment 3. We used ANOVA (PROC GLM) to
analyse the data for trials 1 and 2 of experiment 2 and for
experiment 4 (experimental and control colonies were
analysed separately). For experiment 2, behaviour (nursing versus foraging) and age (7 versus 21 days) were
analysed as fixed effects. For experiment 4, behaviour
(nursing versus all flight experience groups) was analysed
as a fixed effect. After removing nurses from the model to
allow comparisons between flight experience groups, we
analysed the effects of treatment (control, partial restriction, total restriction) and days of foraging (1, 7, 14). For
the effect of days of foraging, we also calculated power
based on observed treatment means and sample sizes
using SAS PROC MIXED (Stroup 2002). For pooled,
experiment-wide analyses (experiments 1, 2 and 4), we
performed nested ANOVAs with trial analysed as a random
effect using PROC MIXED, and with bee nested within
trial. For all post hoc comparisons, P values were adjusted
for experiment-wise error using a Tukey’s adjustment.
RESULTS
Foragers had significantly lower lipid levels than nurse
bees (unpaired two-tailed t tests: trial 1: t35 Z 3.80,
3
2
*
**
1
Trial 1
Trial 2
Figure 2. Lipid levels (means G SE) of foragers (-) and nurses (
from two typical field colonies. *P ! 0.01; **P ! 0.001.
P Z 0.0006; trial 2: t34 Z 2.71, P Z 0.01; Fig. 2). A
pooled analysis of both trials showed a significant effect
of behaviour (nested ANOVA: F1,71.3 Z 18.44, P ! 0.0001)
and no difference between trials (Wald statistic: Z Z 0.24,
P Z 0.41). Furthermore, Oil Red O staining of the fat
bodies revealed markedly less red-stained (lipid-containing) tissue in foragers (Fig. 3a, b) than in nurses (Fig. 3c, d).
The difference in lipid-staining intensity was statistically
significant, with a significantly lower number of counted
red pixels in forager abdominal tissue (unpaired two-tailed
t test: trial 1: t18 Z 5.23, P ! 0.0001; Fig. 3e).
Experiment 2: Are Abdominal Lipid Stores
Associated with Age or Task Performance?
In all three trials, lipid levels in both precocious foragers
and normal-age foragers were lower than those of both
groups of nurses (normal-age and overage). In all trials,
there were significant differences in lipid levels due to
behaviour (nursing versus foraging) (ANOVA: trial 1:
F1,72 Z 12.05, P Z 0.0009, N Z 76; trial 2: F1,41 Z 12.34,
P ! 0.001, N Z 45; unpaired two-tailed t test: trial 3:
t16 Z 3.06, P Z 0.008; Fig. 4). The effect of age (7 versus
21 days) was significant in trial 1 (F1,72 Z 6.53, P Z 0.01),
but not significant in trial 2 (F1,41 Z 0.02, P Z 0.90). There
was no behaviour)age interaction in either trial (trial 1:
F1,72 Z 1.68, P Z 0.20; trial 2: F1,41 Z 0.13, P Z 0.72). The
significant age effect in trial 1 can be attributed to the fact
that lipid levels in precocious foragers were higher in trial
1 than in the other two trials. In a pooled analysis of the
three trials, there were significant differences in abdominal lipid levels due to behaviour (nested ANOVA:
F1,134 Z 32.54, P ! 0.0001), but not age (F1,134 Z 1.33,
P Z 0.25) or trial (Wald statistic: Z Z 0.69, P Z 0.25). The
results of this experiment indicate that differences in
abdominal lipid stores were strongly associated with
differences in bee behaviour and not age.
Experiment 3: Do Abdominal Lipid Stores
Vary with Foraging Experience?
Experiment 1: Do Abdominal Lipid Stores
of Nurses and Foragers Differ?
Lipid (mg)
430
)
To examine the effectiveness of our flight restriction
treatments (unrestricted, partially restricted, totally restricted), we used wing wear as an indicator of flight
experience and sampled bees (N Z 19 for each group) after
7 days of flight (for unrestricted and partially restricted
bees). The unrestricted flight group had the highest proportion of bees with noticeable wing wear (21%), followed
by the bees with partially restricted flight (10.5%). None of
the bees in the totally restricted flight group showed
noticeable wing wear. Although flight activity of each
group of bees was not quantified, casual observations
(A.L.T.) showed a steady stream of foragers throughout the
morning and afternoon, with partially restricted bees
‘waiting’ to exit each day when the second screen was
removed. We can safely conclude that our treatments
caused partial and total restriction of flight.
Foragers in all three flight groups regardless of the
extent of flight experience, had low, forager-typical, lipid
levels (Fig. 5). Nurse bees had significantly higher lipid
TOTH & ROBINSON: NUTRITION AND DIVISION OF LABOUR
(a)
(c)
(b)
(d)
Red pixels (%)
9
(e)
**
6
3
Foragers
Nurses
Figure 3. Representative examples of fat body stained with Oil Red O from two foragers (a, b) and two nurses (c, d) collected from typical field
colonies. Lipid granules are stained red (darkly coloured). (e) Percentage of fat body stained red with Oil Red O in honeybee foragers and
nurses. **P ! 0.001.
levels than all three flight groups (ANOVA: F1,103 Z 16.85,
N Z 127, P ! 0.0001). There was a significant, but much
smaller, difference in lipid levels between the three flight
groups (ANOVA: F2,103 Z 3.68, N Z 112, P Z 0.029), suggesting a possible effect of flight experience. There was no
significant difference in lipid levels as a function of the
number of days of foraging (F2,103 Z 3.68, 2.12,
P Z 0.125). However, because the magnitude of the
difference between mean lipid levels for days 1, 7 and 14
was small, the power of this test was low (38.7% power, or
61.3% probability of type II error). Thus, we cannot rule
out the possibility that there may have been a small but
statistically undetectable effect of days of foraging experience on lipid levels. None the less, lipid levels of bees
with little or no flight experience (bees collected on day 1
of flight experience) were substantially lower than those
of nurse bees.
In the control experiment, we detected no side effects of
the tagging treatment on bee lipid levels. Lipid levels in
free-flying tagged bees were not significantly different
from those of untagged, paint-marked bees (ANOVA:
F1,54 Z 0.04, N Z 58, P Z 0.841). In addition, as in the
previous case, there was no difference in lipid levels as
a function of number of days of foraging (F2,54 Z 0.01,
P Z 0.987).
Experiment 4: Is Behavioural Reversion
Associated with an Increase in Abdominal
Lipid?
Bees did not regain their lipid stores when reverting to
nursing behaviour, at least not within the first 5 days. In
both trials, we found no difference between lipid levels of
431
ANIMAL BEHAVIOUR, 69, 2
2
b'
b'
2
a
a
a
1
a'
**
a'
b
1.5
Age (days)
7
21
7
21
7
Trial 1
Trial 2
Trial 3
Figure 4. Lipid levels (means G SE) of precocious foragers (-) and
normal-age nurses ( ) collected on day 7, and normal-age foragers
(G) and overage nurses (,) collected on day 21 from single-cohort
colonies. Lipid levels that were significantly different (within trials)
are designated with different letters (a, b, a0 , b0 ). **P ! 0.001.
reverted nurses and foragers (unpaired two-tailed t tests:
trial 1: t35 Z 1.71, P Z 0.095; trial 2: t37 Z 0.72,
P Z 0.48; Fig. 6). A pooled analysis gave similar results
(nested ANOVA: F1,74 Z 0.15, P Z 0.701) with no difference between trials (Wald statistic: Z Z 0.76, P Z 0.22).
Bees in both groups had low, forager-typical lipid levels.
DISCUSSION
The results of this study show a strong association
between nutrition and division of labour in honeybees.
Foragers typically had half the lipid stores of nurses,
paralleling the lean forager–corpulent nest worker pattern
observed in other social insects. For example, in the ant
Pogonomyrmex owyheei, foraging workers are 40% leaner
than nest workers (Porter & Jorgensen 1981). In another
species of this genus, Pogonomyrmex badius, young workers
at the bottom of a nest have 30% fat, older workers near
the top of a nest have less (w23%), and foragers always
have less than 10% (Tschinkel 1998). Callow workers
of Prenolepis imparis ants act as ‘corpulents’, with 60%
abdominal fat; the following year they lose this fat
almost entirely after brood rearing and become foragers
(Tschinkel 1987).
2
c
1.5
b
1
ab
a
ab ab
ab ab
a
a
0.5
1
7
14
Nurse
Collection day
Figure 5. Lipid levels (means G SE) of bees with unrestricted flight
(-), partially restricted flight ( ) and totally restricted flight (G)
collected on the first, seventh and 14th day following the onset of
foraging by unrestricted and partially restricted bees, and lipid levels
of nurses collected from the same colony. Lipid levels that were
significantly different are designated with different letters (a, b, c).
Lipid (mg)
Lipid (mg)
3
Lipid (mg)
432
1
NS
NS
0.5
Trial 1
Trial 2
Figure 6. Lipid levels (means G SE) of honeybee foragers (-) and
reverted nurses ( ).
Results from our single-cohort colony experiment show
that nutritional differences are more closely related to
behavioural state than to age. Overage nurses delayed
foraging, but maintained high lipid levels. Conversely,
bees that foraged precociously had low lipid levels despite
their young age. These results suggest that changes in lipid
stores are mediated by changes in lipid metabolism or
feeding behaviour that are associated with some of the
different tasks performed by a worker honeybee during
her life.
Because there are genotypic differences in foraging
ontogeny in honeybees (e.g. Giray et al. 1999), the
foragers we sampled from the single-cohort colonies may
have shown a genotypic predisposition to early foraging.
Thus, the differences in lipid levels seen in precocious
foragers may have been a consequence of genotypic
variation in lipid physiology unrelated to foraging. However, we consider this improbable because we obtained
consistent results from three separate trials, each performed with bees of a different mix of genotypes, and
thus, it is unlikely that the observed lipid differences could
be attributed to repeated coincidences of the occurrence of
early foraging genotypes with low-lipid genotypes. Furthermore, in all experiments we observed patterns of lipid
levels that reflect behaviour and not age. There may well
be genotypic differences in lipid physiology in honeybees,
but this is not likely to confound our interpretation of the
results.
Results of experiment 3 suggest that foraging itself does
not have a strong effect on lipid levels. The treatment
effectively limited foraging activity, and a control experiment showed there was no additional lipid depletion
from carrying a tag. In both experimental and control
colonies, there was no detectable effect of days of foraging
experience on lipid levels. Although experimental restriction of flight had a significant effect on lipid levels, this
effect was small (on the order of tenths of a milligram of
lipid) as opposed to the large difference (nearly 1 mg)
found between foragers and nurses. Therefore, our results
suggest slight lipid depletion may occur during foraging.
However, lipid levels of foragers were already substantially
lower than those of nurses on the first day of foraging.
Furthermore, even bees that never made a single flight
TOTH & ROBINSON: NUTRITION AND DIVISION OF LABOUR
(totally restricted bees collected on day 1) had low lipid
levels at an age when partially restricted and unrestricted
bees foraged. Although only one trial of this experiment
was performed, these results indicate that a substantial
amount of lipid depletion can occur prior to foraging. This
suggests that lipid stores are mainly metabolized during
nest work activities such as nursing, feeding nestmates, or
producing wax, perhaps in anticipation of the onset of
foraging. Lipid depletion prior to foraging has also been
observed in the wasp Polybia occidentalis (O’Donnell &
Jeanne 1995).
Our results suggest that honeybees rely on carbohydrates (not stored lipid) for energy when foraging. This is
consistent with the finding that honeybee and Vespula
vulgaris wasp foragers cease flight and quickly die after the
crop is empty (Lorenz et al. 2001). In the migratory locust,
Locusta migratoria, the initial 20–30 min of flight are
fuelled by the disaccharide trehalose, with a switch to
metabolism of lipid stores to sustain prolonged flight
(Weis-Fogh 1952; Goldsworthy et al. 1979). In honeybees,
foraging flights last about 10 min for pollen and
30–80 min for nectar (Winston 1987). If foragers use
energy obtained from nectar/honey ingested in the hive
(plus a portion of collected nectar to fuel return flights), it
is feasible that honeybees are able to sustain most foraging
flights from recently ingested carbohydrates.
In contrast to foraging, there does not appear to be as
close an association between levels of abdominal lipid
stores and nursing behaviour in honeybees. We found
that bees that reverted to nursing did not regain their lipid
stores, but we do not know how active these bees were at
nursing. We did notice substantial mortality of larvae
cared for by reverted nurses in the reversion colonies
(A.L.T., personal observation), consistent with the results
of a previous study (Robinson et al. 1992). This observation suggests that reverted nurses may be less effective,
due either to physiological and/or behavioural deficits.
The limited lipid stores of reverted nurses may result in
a lower quality and/or quantity of brood food produced by
the hypopharyngeal glands, leading to reduced nursing
ability. This suggestion is consistent with recent findings
that the lipoprotein vitellogenin, which is produced by
the fat body, is involved in brood food production by
worker bees (Amdam et al. 2003).
Although our experiments addressed one component of
nutritional state (lipid stores), the fat body is also an
important site of storage of protein reserves. Paralleling
our results, foragers also show low protein content in fat
body cells (Fluri & Bogdanov 1987), and synthesis of the
storage protein vitellogenin is shut down (Pinto et al.
2000). The consumption of pollen, bees’ only dietary
source of fat and amino acids, is extremely high in nurse
bees and minimal in foragers (Crailsheim et al. 1992). It is
likely that protein-based and carbohydrate-based nutritional patterns are closely linked since vitellogenin, the
most abundant storage protein in female insects, is
a lipoprotein whose synthesis depends on amino acid
and lipid reserves (Chapman 1998).
Our finding of nutritional depletion prior to foraging is
consistent with the possibility that nutritional depletion
can influence the onset of foraging behaviour. Nutrition
could affect worker behavioural development through
food input (changes in the quality and quantity of the
diet), intrinsic physiological changes (e.g. metabolic and
neuroendocrine shifts), and/or social input (food exchange). Adult honeybees switch from high consumption
of pollen during the first weeks of life to a mostly
carbohydrate-based diet later in life as foragers (Crailsheim
et al. 1992). This dietary change could potentially affect
the behavioural and physiological shifts that accompany
worker honeybee behavioural development. For example,
nutrient depletion in honeybees might lead to an increase
in circulating titres of juvenile hormone (Kaatz et al. 1994;
Pinto et al. 2000), and an increase in titres of this
hormone is associated with the onset of foraging (reviewed by Bloch et al. 2002). Starvation can cause increased expression of a neuropeptide that induces juvenile
hormone production in Manduca sexta larvae (Bhaskaran
& Jones 1980; Lee & Horodyski 2002). Starvation also
causes an increase in octopamine in some insects
(Hirashima et al. 1992), and this neurochemical has also
been shown to influence the onset of foraging in honeybees (Schulz & Robinson 2001). However, to evaluate the
possibility that nutrition affects division of labour, it is
necessary to go beyond the correlations reported here and
determine whether experimental depletion of lipid stores
can actually cause an earlier onset of foraging.
The observed changes in worker nutrition may be
related to the regulation of division of labour by social
inhibition (Huang & Robinson 1999). Young bees are
inhibited from becoming foragers in part by direct social
contact with older bees (Huang & Robinson 1992, 1996;
Huang et al. 1998). Because one of the primary forms of
social contact in honeybees is trophallaxis (social food
exchange), a connection may exist between trophallaxis,
nutrition and social inhibition. Blanchard et al. (2000)
suggested that the ‘unidirectional food flow’ from foragers
to nurses (Korst & Velthuis 1982; Leibig et al. 1997) may
contribute to the lean forager–corpulent nest worker
pattern found in many social insects. If this is the case,
then food exchange between foragers that tend to donate
food and young bees that tend to receive food (Free 1955)
could lead to an enhanced nutritional state in young bees,
and perhaps inhibition of foraging behaviour. We suggest
that a thorough investigation of the connection between
social interactions and nutrition could have important
implications for the social inhibition model.
The ‘lean forager–corpulent nest worker’ pattern has
been observed numerous times in the literature (see
Introduction). Thus, there must be strong selective pressure causing repeated convergence on this pattern across
taxa of social insects. Porter & Jorgensen (1981) suggested
that foragers serve as a ‘disposable caste’ for the colony.
They argue that since foraging is the most energetically
costly and dangerous task a worker can perform, it is
reserved until the last portion of a worker’s life. The
disposable caste idea has implications for colony organization as well as worker ergonomics (Porter & Jorgensen
1981; O’Donnell & Jeanne 1995). By reserving tasks that
incur high mortality risk for the most nutritionally depleted workers, the amount of energy lost by the colony is
minimized. From the perspective of foraging energetics,
433
434
ANIMAL BEHAVIOUR, 69, 2
lean foragers save energy by having less weight (e.g. fat
stores) to carry on foraging trips. Blanchard et al. (2000)
also pointed out that the reduction of nutritional reserves
in the abdomen, especially by limiting the volume of the
fat body, leaves more room for foragers to store liquid food
in the crop.
As a corollary to the disposable forager theory, one can
also consider the adaptive value of the corpulent nest
worker. Nest workers may function as living vessels for the
storage of colony energy (Porter & Jorgensen 1981). This
occurs to an extreme degree in some ants, in which replete
workers remain in the nest and store huge amounts of
liquid food in their crops (Wilson 1971). ‘Fat body
repletes’ have also been found in several species of ants,
in which lipid stores in the fat body are hypertrophied to
an extreme degree (Burgett & Young 1974; Tschinkel
1987; Borgesen 2000). In cases such as these, but also in
less extreme cases such as honeybees, fat nest workers may
serve as a valuable sink of colony energy.
This work provides a first step into the study of
nutritional influences on patterns of worker division of
labour. Further studies of the physiological and molecular
pathways underlying nutritional changes in worker honeybees can improve our understanding of the mechanisms
of honeybee division of labour, via interactions with other
known factors such as juvenile hormone titres, octopamine levels and gene expression patterns (Ben-Shahar
et al. 2002; Robinson 2002; Whitfield et al. 2003).
Furthermore, there is a need for parallel studies in other
social insects to understand the evolutionary significance
of this pattern in a comparative context. Additional
studies of worker nutrition have the potential to provide
important new insights into both the mechanisms and
evolution of division of labour in the social insects.
Acknowledgments
We thank K. Pruiett for expert assistance with the bees;
M. Sakashita for dissections and lipid analyses; S. E.
Fahrbach and R. A. Velarde for advice on histology and
lipid assays; F. E. Miguez for assistance with statistics; and
J. L. Beverly, S. A. Cameron, S. E. Fahrbach and members of
the Robinson laboratory for comments that improved the
manuscript. Supported by Clark Research Grant, Program
in Ecology and Evolutionary Biology Research Grants from
SIB/PEEB to A.L.T. and grants from the National Science
Foundation, the Burroughs–Wellcome Trust, and the University of Illinois Research Board to G.E.R.
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