Flight performance of pollen
starved honey bees and
incomplete compensation
through ingestion after early life
pollen deprivation
Robert Brodschneider
1
*, Eslam Omar
1
,
2
and Karl Crailsheim
1
1
University of Graz, Institute of Biology, Graz, Austria,
2
Plant Protection Department, Facu lty of
Agriculture, Assiut University, Assiut, Egypt
We investigated the effect of adult honey bee pollen nutrition on the flight
performance of honey bees. Therefore, caged bees were allowed to perform
30 min of defecation/training flights every second day before flight
performance of pollen-fed bees and pollen-deprived bees older than
16 days were compared in a flight mill. We first fed 10 µL of 1 M glucose
solution to bees, and after they metabolized this during flight, they were fed
10 µL of 2 M glucose solution for a second flight test. Pollen-deprived bees flew
longer and further than pollen-fed bees in both flights. Pollen-fed bees flew
faster in the early period at the beginning of flights, whereas pollen-deprived
bees were faster in the final phases. Pollen-fed bees were able to raise their
maximum flight speed in 2 M glucose solution flights, whereas pollen-
constraint bees were not. The two groups did not differ in abdomen fresh
weight, but the fresh weight of the head and thorax and dry weight of the head,
thorax and abdomen were higher in pollen-fed bees. In a second experiment,
we constrained pollen consumption of caged bees during the first 7 days and
compared daily consumption of bees from day 8–16 to consumption of bees
unrestricted in pollen. We found that pollen-deprived bees perceive the pollen
shortage and try to compensate for their needs by consuming significantly
more pollen at the later phase of their life than pollen-fed bees of the same age.
Still, bees constrained from pollen in the first 7 days did only reach 51.1% of the
lifetime consumption of unconstrained bees. This shows that bees can sense
the need for essential nutrients from pollen, but their physiological apparatus
does not allow them to fully compensate for their early life constraint. Pollen
deprivation only in the first 7 days of worker life likewise significantly reduced
fresh and dry weights of the body sections (head, thorax, and abdomen) and
survival. This underlines the importance of protein consumption in a short
critical period early in adult bees’ lives for their development and their
performance later in life.
KEYWORDS
flight musculature, protein, consumption, thorax, flight calorimeter, Apis mellifera,
nutrition
OPEN ACCESS
EDITED BY
Priyadarshini Chakrabarti Basu,
Oregon State University, United States
REVIEWED BY
Hussain Ali,
Agricultural Research Institute Tarnab
Peshawar, Pakistan
Hamzeh Izadi,
Vali-E-Asr University of Rafsanjan, Iran
Ramesh Sagili,
Oregon State University, United States
*CORRESPONDENCE
Robert Brodschneider,
SPECIALTY SECTION
This article was submitted to
Invertebrate Physiology,
a section of the journal
Frontiers in Physiology
RECEIVED 26 July 2022
ACCEPTED 21 November 2022
PUBLISHED 09 December 2022
CITATION
Brodschneider R, Omar E and
Crailsheim K (2022), Flight performance
of pollen starved honey bees and
incomplete compensation through
ingestion after early life
pollen deprivation.
Front. Physiol. 13:1004150.
doi: 10.3389/fphys.2022.1004150
COPYRIGHT
© 2022 Brodschneider, Omar and
Crailsheim. This is an open-access
article distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms.
Frontiers in Physiology frontiersin.org01
TYPE Original Research
PUBLISHED 09 December 2022
DOI 10.3389/fphys.2022.1004150
1 Introduction
The main natural food sources of honey bees are nectar (or
honeydew) and pollen (Brodschneider and Crailsheim, 2010).
The two liquid carbohydrate sources provide bees with energy
which they need for survival, thermoregulation and flight
metabolism. Pollen supplies bees with proteins, lipids,
minerals, and vitamins and is needed for brood rearing and
adult growth (Schmidt and Buchmann, 1985; Brodschneider and
Crailsheim, 2010). Seasonal variations in pollen supply can lead
to different nutritive values of the diet for bees, whereby
polyfloral diets comprised of the pollen of different flowers
are beneficial for honey bee health (di Pasquale et al., 2013;
Frias et al., 2016; Omar et al., 2017). De Groot (1953) identified
ten essential amino acids that bees need for maximum gain of
body mass. Later, Hendriksma et al. (2019) found that nutrition
with these essential amino acids is important for developing flight
muscles in caged and colony bees. Adult bees, in a colony or
cages, consume pollen mainly in the first days after emergence
(Crailsheim and Stolberg, 1989; Crailsheim et al., 1992; Omar
et al., 2017) and if pollen quality is low, they reach their
maximum thorax masses late (Hagedorn and Moeller, 1968).
Fernandez-Winckler and da Cruz-Landim (2008) studied the
ultrastructural development of flight muscles in workers of two
species of eusocial bees (Apis mellifera and Scaptotrigona postica ).
In both species, workers emerge with immature flight muscles
and complete their development during the nurse bee stage. The
changes during the development of the thorax include synthesis
of high numbers of myofibrils, mitochondria and many enzymes
for carbohydrate catabolism located in flight muscles (Herold,
1965; Hersch et al., 1978; Suarez, 2000). During flight, honey bees
increase their metabolic rate to the top level (Nachtigall et al.,
1995; Harrison and Fewell, 2002). Bees fuel the flight almost
exclusively with carbohydrates from their honey stomach. Body
reserves do not play an important role for flight, as adult bees
have comparable low glycogen stores (Gmeinbauer and
Crailsheim, 1993; Hrassnigg and Crailsheim, 2005
; Hrassnigg
et al., 2005). When in need of energy before foraging flights,
workers provision themselves with sugars from honey, which
they obtain from honey stores or via trophallactic contacts
(Crailsheim, 1998; Tan et al., 2015).
The consumption of pollen by an individual honey bee
worker strongly depends on the age and activities in the
colonies. As pointed out before, bees start feeding on beebread
soon after they emerge, whereas older bees cease consumption.
Young bees are also provided protein-rich jelly from nurse bees
by trophallaxis. The high protein turnover of young to middle-
aged bees is closely connected to the brood-caring behavior of
nurse bees with highly developed hypopharyngeal glands (Dietz,
1969; Crailsheim et al., 1992; Crailsheim, 1998; Amdam et al.,
2009). To obtain the proteins from pollen, the midgut of nurse
bees has a high proteolytic activity and this enzymatic activity
decreases in older foragers, which in turn produce high amounts
of carbohydrate digesting enzymes in their hypopharyngeal
glands (Moritz and Crailsheim, 1987; Crailsheim et al., 1992;
Ohashi et al., 1999). These enzymes accumulate in the midgut
and break down even complex sugars. In addition, the microbial
community of the midgut and pollen enzymes can contribute to
saccharolytic potential (Ricigliano et al., 2017).
As a result of the pollen feeding period early in adult life,
protein content and dry weight increase in young bees, and again
slightly decrease in older bees (De Groot, 1953). When protein
consumption is inadequate, honey bee worker longevity, colony
brood area and honey production are reduced. To adapt to
environmental pollen shortage, bees in colonies first finish the
stored beebread and later exhaust their body reserves to retain
brood rearing for a short time before they start cannibalizing
larvae ( Haydak, 1935; Brodschneider and Crailsheim, 2010).
Crailsheim and Stolberg (1989) compared changes in the dry
weight of caged and free-flying bees. They found that in free-
flying bees dry weight increased until the age of 3 days and
remarkably decreased later. In caged bees, dry weight continued
to increase until the age of 8 days, probably because caged bees
cannot defecate undigested materials. The dry weight of caged
bees was highest in bees fed with honey and beebread compared
to several other in vitro diets. The dry weight of bees deprived of
protein in cages for 3 days and then transferred to colonies did
not differ much from hive controls, suggesting some ability to
compensate for early pollen starvation.Early developmental
nutrition profoundly influences phenotypic trajectories and
affects adult physiology, behavior, longevity, and fitness in
many animal species (Buchanan et al., 2022). For bees,
Crailsheim and Stolberg (1989) investigated the effect of
delaying the protein nutrition on the levels of proteolytic
activities and the size of the hypopharyngeal glands. Still,
nothing is known about the perception, attempts and abilities
of bees compensating for pollen starvation during this critical
period early in adult life by feeding more pollen later in life.
Therefore we studied, if bees are 1) sensing a nutritional
deficiency experienced in early adult life (which could
optionally, but not necessarily, result in an increased
consumption later in life) and 2) if such an experimentally
induced delayed consumption is successful in satisfying the
bees’ lifetime protein intake. As the early starvation and
possible compensation later in life might be too late for
showing an effect on the development of hypopharyngeal
glands needed at young age, we measured thorax weight as an
indicator for nutrient assimilation and flight muscles
(Hendriksma et al., 2019; Ricigliano and Simone-Finstrom,
2020; Ricigliano et al., 2022). Regarding macronutrient ratios,
social insects do have astounding abilities to anticipate and react
towards certain intake goals (Simpson and Raubenheimer, 2009;
Vaudo et al., 2016). Bees may even be able to select a certain
forage source that complements another available diet which is
deficient in a particular amino acid (Hendriksma and Shafir,
2016). However, the ability of bees of adjusting delayed food
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Brodschneider et al. 10.3389/fphys.2022.1004150
intake after pollen starvation during early life has not been
studied before. We designed an experiment to measure
dynamics of pollen consumption of bees unrestricted in pollen
and constrained to delayed pollen consumption. The daily pollen
ingestion of bees in this experiment informs us if bees with a
delayed consumption attempt to compensate for early pollen
starvation. If they do, lifetime consumption informs us, if they
can fully compensate this deficit.
Larval diet quality has been shown to affect adult worker
bees’ flight performance (Brodschneider et al., 2009) and flight
onset in drones (Metz and Tarpy, 2022). Here we investigate if
adult protein nutrition as well affects flight muscle development
and flight ability of the honey bee. We, therefore developed a test
in which we could control adult bee protein nutrition in cages but
allow bees to defecate and develop their flight apparatus in
‘training flights’ outside the cage. Our second aim was to
study if caged honey bees, deprived of pollen during the first
days of life, would attempt to compensate for their needs in a later
life phase, and if they can satisfy their protein needs then.
2 Materials and methods
2.1 Honey bees and pollen diet
Honey bee workers were obtained by incubating sealed brood
combs of different, unrelated Apis mellifera carnica colonies at
34.5
°
C under standard conditions (Williams et al., 2013; Omar
et al., 2017). Each comb was placed in a separate comb cage. Newly
emerged bees (0–24 h old) from these brood combs were mixed
and randomly introduced into adult bee cages. Two different types
of cages were used in the two different experiments, see below for
specifications. Pollen loads for preparing pollen diet were collected
using front-mounted plastic pollen traps (Anel, Greece) on several
days from colonies at the same apiary and frozen at -20
°
C. A
homogeneous mixture of this pollen of several colonies and
undefined polyfloral botanical origin was made and was
kneaded with 10% (w/w) water until a homogeneous pollen
dough was formed (Williams et al., 2013). The dough was
further stored at -20
°
C and thawed on the day of use. The
pollen diet was presented in one-half of a cylindrical 10 ml
plastic tube. Standard carbohydrate feeding (50% w/v sucrose
solution) was provided ad libitum in 1.5 ml punctured
Eppendorf vials and renewed daily.
2.2 Experiment 1: Effect of adult nutrition
on flight performance
For this experiment we used wooden cages (15 × 15 × 5.5 cm)
covered with a wooden board from one side, which could be
opened to remove bees and change diets. The other side was
covered with a grid that allowed air ventilation. Each cage was
provided with a piece of wax for bees to cluster on. Four cages
were used in this experiment, each cage containing 60 newly
emerged honey bees. The four cages were divided into two groups
with two replicates each and kept in an incubator at 34.5
°
C. The
two cages from the first group received sugar solution as
described above and pollen diet ad libitum during the first
16 days of the experiment (pollen-fed bees). The pollen diet
was renewed every day. The two cages from the second group
received sugar solution only (pollen-deprived bees).
2.2.1 Flight training
From day three on and every 2 days thereafter, we allowed
bees from both groups separately to fly free and defecate for
30 min in a 30 × 30 × 60 cm glass box (Figure 1). On one side of
the glass box a light trap attracted bees to fly to the light and from
the other side it could be closed by a net to prevent the flying bees
from escaping. After the flight training of the first group was
finished, bees were collected by a modified hand-held vacuum
cleaner, by hand or with forceps and put back in their belonging
cages, before the next group of bees was released for flight
training. This way, the two groups of bees could not be mixed
up, and no marking of bees was necessary.
FIGURE 1
Glass box used for flight training. The box was covered with
mosquito net (meshed) and light trap (yellow) on the back. Arrow
indicates where bees are introduced.
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2.2.2 Flight experiments
We tested the flight performance of caged bees following the
protocols in Brodschneider et al. (2009) and Scheiner et al.
(2013). We started flight experiments when the caged bees
reached the age of 16 days and continued daily until day 29.
Alternatingly, one bee from the group of pollen-deprived bees
followed by a pollen-fed bee was taken for flight experiments.
Each bee was attached by a small tube on the thorax to the
14.5 cm long arm of a flight mill (roundabout), so one rotation
covered 91.06 cm. The attached bee was repeatedly stimulated to
begin the flight by removing a small ball of paper that the bee held
with her legs. Workers that did not start a continuous flight
within 20 min were noted down for calculating the rate of (un-)
successful flyers and discarded.
After an emptying flight which forces the bee to spend
most of its sugar reserves from the gut and the hemolymph,
the bee was fed 10 μL of 1 M glucos e solution with a micro-
pipette and given a resting time of exactly 5 minutes. Be fore
and after feeding, bees were weighed on an analytical balance
to the nearest 0.1 mg, to confirm complete ingestion of the
glucose solution. Then the bee was stimulated to flyandempty
all i ts sugar reserves (first flight,1M).Thenumberof
rotations, duration per rotation flown by the bee in the
flight mill was recorded by a computer, and the overall
flight time was additionally clocked by hand, so that only
the active flight period was considered in further calculations.
After the be e ran ou t of ene rgy (defined as not being able to
move the arm of the flight mill any more) the same bee
received 10 μLof2Mglucosesolution(secondflight, 2 M)
and the fl ight procedure was repeated as above.
Because ambient temperature affects flight metabolic rate
and speed (Hrassnigg and Crailsheim, 1999), the temperature in
the flight mill was measured in real time and automatically
logged for every rotation of the flight mill. Temperature was
manually maintained at around 26
°
C using a 40W light bulb in a
lamp above the flight mill. This lamp could be placed closer to
increase, or further away from the flight mill to decrease the
temperature.
Flight distance, average speed, maximum speed per minute
and mean metabolic power were calculated from the number of
rounds logged by the flight mill and the radius of the flight mill,
the active flight time, the amount of glucose in feedings and the
food energy in glucose (15.7kJ per g) (Gmeinbauer and
Crailsheim, 1993; Brodschneider et al., 2009). From the flight
mill log data, the maximum speed per round was extracted for
each flight. We calculated average speed for different periods of
the flights, which were cut into three periods (10 min each) for
1M flights, or four periods (10 min each, except the last one,
which was 15 min) for 2 M flights.
After the two flights, each bee was dissected into the head,
thorax (including legs and wings), and abdomen, and each part
was weighed to the nearest 0.1 mg. The sections were dried for
7 days in an incubator at 70
°
C and dry weight was measured to
the nearest 0.1 mg (Human et al., 2013).
2.3 Experiment 2: Compensation of early
pollen deprivation
The experimental cages in this experiment consisted of clear
0.3 L plastic cups with two holes, one in the base of the cup and
the other on the side of the cup. The cages were supplied with a
wax comb. The cage was closed from below by a grid that allowed
air to pass through (Evans et al., 2009). After we placed bees into
the cage, we covered the hole in the base with a tape. The other
hole was closed with a 1.5 ml Eppendorf vial in which three small
holes had been punctured from which the bees could drink
sucrose solution. The pollen diet was presented in one-half of a
cylindrical 10 ml plastic tube, as in the previous experiment. All
bees in cages were kept in the dark in an incubator at 34.5
°
C for
16 days.
Six hundred newly emerged honey bees younger than 24 h
from the same origin as explained above were randomly
distributed into twelve cages, 50 bees per cage. The twelve
cages were divided into two groups of six replicates each. The
first group received pollen diet from day one until day 16 (‘full
access’). The second group did not receive pollen in the first
7 days, but from day eight until day 16 (‘constraint access’).
Sucrose solution was applied ad libitum and renewed daily. Dead
bees were counted daily and removed from the cages. Every day
we renewed the pollen diet. To measure the amount consumed,
the difference between the weight of diet applied and the weight
after bees consumed was measured on an analytical balance to
the nearest 0.1 mg. To calculate food consumption per bee, dead
bees were assumed not to have consumed any food since the
prior food was changed. The period during which bees had access
to the protein diet was measured to the minute but was
recalculated to reflect 24 h consumption (Williams et al., 2013;
Omar et al., 2017). We calculated cumulative consumption per
bee for the group always allowed feeding on pollen (‘full access,
day 1–16’), and additionally for the second phase of this
experiment only (‘full access, day 8–16’, ignoring the
consumption on the first 7 days). This allowed comparing the
normal consumption of bees older than 8 days (‘full access, day
8–16’) to bees given access to pollen only on days 8–16
(‘constraint access, day 8–16’). Cumulative consumption per
bee on day 16 represents the lifetime consumption of bees in
this experiment and informs us if bees with a delayed
consumption attempt to and are able to compensate early life
pollen starvation.
Dead bees were removed and counted daily to analyze
survival. After 16 days, the fresh and dry weight of body
sections of ten randomly chosen bees from each cage were
measured the same as in the first experiment.
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2.4 Statistics
We compared the rate of successful flights of pollen-fed and
pollen-deprived bees with two-sided Pearson’s chi-square test.
Age of bees used for flight experiments and all flight parameters
(duration, distance, average and maximum speed, ambient
temperature) followed a normal distribution (one-sample
Kolmogorov-Smirnov test, p > 0.07) with unequal variances in
some variables (Levene statistic, p = 0.00–0.95). We therefore
compared groups using two-tailed Student’s t-test assuming or
not assuming equal variances, where applicable. First and second
flights of same bees were compared with two-tailed paired-
samples Student’s t-tests.
Fresh and dry weight of body sections followed a normal
distribution (one-sample Kolmogorov-Smirnov test, p > 0.1)
with unequal variances in some variables (Levene statistic, p =
0.02–0.73). We therefore compared groups using two-tailed
Student’s t-tests assuming or not assuming equal variances,
where applicable.
Consumption data of experiment 2 (compensation of early
pollen deprivation) followed a normal distribution (one-sample
Kolmogorov-Smirnov test, p > 0.7) with homogeneity of
variances (Levene statistic, p > 0.3) and was therefore
compared using one-way ANOVA with Bonferroni correction
for multiple comparisons. Daily pollen consumptions per bee on
each of day eight to 16 followed a normal distribution (one-
sample Kolmogorov-Smirnov test, p > 0.3) with unequal
variances in some variables (Levene statistic, p = 0.04–0.82)
and were compared using two-tailed Student’s t-test assuming
or not assuming equal variances, where applicable. We compared
survival of bees with full and constraint consumption with
Mantel-Cox log-rank tests. All statistical analyses were made
in SPSS Statistics version 21 (IBM).
3 Results
3.1 Experiment 1: Effect of adult nutrition
on flight performance
Of all tested bees, 83.6% were capable to flyintheflight mill.
88.5% (23 out of 26) of bees that received a pollen diet successfully
TABLE 1 Flight parameters of pollen-fed bees and pollen-deprived bees in two feeding regimes (10 μL of 1 M glucose solution, 10 μL of 2 M glucose
solution).
Feeding regime Pollen-fed bees Pollen-deprived bees p
10 μL 1 M glucose N 23 23
Age (d) 22.8 ± 4.1 22.7 ± 4.3 0.917
Duration (sec.) 1,048.3 ± 205.1 1,308.2 ± 374.2 0.006
Distance (m) 1,063.0 ± 283.6 1,280.4 ± 411.8 0.044
Average speed (m/s) 1.0 ± 0.3 1.0 ± 0.2 0.545
Average speed minute 1 to 10 (m/s) 1.16 ± 0.46 0.95 ± 0.43 <0.001
Average speed minute 11 to 20 (m/s) 1.05 ± 0.4 1.13 ± 0.38 0.029
Average speed minute 21 to 31 (m/s) 0.59 ± 0.2 0.96 ± 0.35 <0.001
Maximum speed per minute (m/s) 1.5 ± 0.4 1.5 ± 0.2 0.838
Maximum speed per round (m/s) 1.6 ± 0.4 1.7 ± 0.5 0.415
Ambient temperature (
°
C) 26.3 ± 0.7 25.9 ± 1.0 0.196
10 μL 2 M glucose N 23 18
Age (d) 22.8 ± 4.1 23 ± 4.7 0.875
Duration (Sec.) 1708.6 ± 299.2 2,622.5 ± 1,131.4 0.003
Distance (m) 2032.6 ± 517.0 2,942.2 ± 1,165.2 0.005
Average speed (m/s) 1.2 ± 0.3 1.2 ± 0.2 0.571
Average speed minute 1 to 10 (m/s) 1.14 ± 0.52 1.06 ± 0.43 0.084
Average speed minute 11 to 20 (m/s) 1.5 ± 0.44 1.22 ± 0.34 <0.001
Average speed minute 21 to 30 (m/s) 1.16 ± 0.44 1.31 ± 0.33 <0.001
Average speed minute 31 to 46 (m/s) 0.84 ± 0.27 1.24 ± 0.37 <0.001
Maximum speed per minute (m/s) 1.6 ± 0.4 1.6 ± 0.3 0.553
Maximum speed per round (m/s) 1.7 ± 0.5 1.6 ± 0.3 0.402
Ambient temperature (
°
C) 26.7 ± 0.6 26.6 ± 0.8 0.847
Means and standard deviations, sample sizes, and p-values (two-tailed Student’s t-tests) are given. For paired comparisons between 1 M and 2 M flights, see results.
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flew in the flight mill, compared to 79.3% (23 out of 29) of bees that
were pollen-deprived. Flight success rates did not differ between the
two groups (Χ
2
= 0.36, n =55,p > 0.05). Five in the first flight
successful bees of the pollen-deprived group failed to flyinthe2M
flight. The age of bees used for flight experiments, just as the
temperature during flight experiments, was successfully
controlled not to differ between the two groups, (p > 0.1,
Student’s t-test, Table 1). Flight data (flight speed for each
minute of the flight, averaged for all individuals) for both groups
and the two different glucose feedings are shown in Figure 2. Pollen-
deprived bees flew longer and further than pollen-fed bees in both
flights (p < 0.05, Student’s t-test, Table 1). Both groups did not differ
in their average speed for the entire period of flight, but the pollen-
fed bees flew significant ly faster in the first 10-minutes-period of
acceleration at the beginning of the 1 M flights (p < 0.001), but not in
2Mflights (p = 0.084, Student’s t-test, Table 1). Pollen-deprived bees
flew significantly faster in the rest of the flight periods of both fligh ts
(second and third periods in the 1 M flight and second, third and
fourth periods in the 2 M fligh t, Table 1).
For both, pollen-fed and pollen-deprived bees, time, distance,
and average speed were higher in 2 M flights than in 1 M
flights
(p < 0.05, paired-samples Student’s t-tests). Maximum speed per
minute and per round of pollen-fed bees was higher in 2 M flights
than in 1 M flights (p < 0.05, paired-samples Student’s t-tests)
whereas we found no differences in maximum speed per minute
or per round between flights with 1 M and 2 M in pollen-
deprived bees (p > 0.05, paired-samples Student’s t-tests).
Average speeds of the first and second period of flights were
also higher in second flights with 2 M glucose feeding compared
to flights with 1 M glucose feeding (p < 0.05, paired-samples
Student’s t-tests), except in 1 M flights of pollen-deprived bees,
where p = 0.070.
Knowing the flight duration and the amount of spent (=fed)
glucose, we calculated the mean (± standard deviation) metabolic
power of honey bee flight (Gmeinbauer and Crailsheim, 1993;
Nachtigall et al., 1995; Brodschneider et al., 2009). This was 28.0 ±
5.9 mW (n = 23) for pollen-fed bees and 23.6 ± 7.6 mW (n =23)for
pollen-deprived bees in 1 M flights . For 2 M glucose solution flights,
the mean metabolic power was 34.0 ± 5.6 mW (n = 23) for pollen-fed
bees and 27.8 ± 19.2 mW (n = 18) for pollen-deprived bees.
Pollen-fed and pollen-deprived bees did not differ in abdomen
fresh weight (p > 0.05, Student’s t-test, Figure 3), but the fresh weight
of head and thorax were significantly higher in pollen-fed bees (p <
0.05, Student’s t-test). Dry weights of the head, thorax and abdomen
were significantly higher in pollen-fed thaninpollen-deprivedbees
(n =24–26, the non-flying bees were also included in weight
measurements, p < 0.05, Student’s t-test, Figure 3).
3.2 Experiment 2: Compensation of early
pollen deprivation
We found significant differences between all cumulative
lifetime consumptions. An individual worker bee in a cage
with full pollen supply cumulatively consumed a mean of
FIGURE 2
Mean flight speed of pollen-fed and pollen-deprived bees. (A)
After being fed 10 μL of 1 M glucose solution (n = 23 bees per
group) and (B) 10 μL of 2 M glucose solution (n = 23 pollen-fed
bees and 18 pollen-deprived bees).
FIGURE 3
Fresh and dry weight of the head, thorax, and abdomen of
pollen-fed bees and pollen-deprived bees from flight
experiments. Means and standard deviations are shown: n =
24–26 bees. * indicates p < 0.05, Student’s t-test.
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86.6 mg in the first 16 days of her life, the late life consumption in
the period from day 8–16 was 25.5 mg (see boxplots for day 16 in
Figure 4 ). Bees prevented from pollen consumption in early life
consumed a mean of 44.3 mg pollen in total in the period from
day 8 to day 16. The cumulative lifetime consumption of bees
with full pollen supply (‘full access, day 1 –16’), bees allowed
feeding on pollen only after day eight (‘constraint access, day
8–16’) and the amount that unrestricted bees consumed in the
second half of the experiment (‘full access, days 8–16’) were all
significantly different from each other (n = 6 each, p < 0.001, one-
way ANOVA with Bonferroni multiple comparisons post-hoc
test, Figure 4). Also, daily pollen intakes after day 8 were
significantly higher in bees not allowed feeding pollen for the
first 7 days compared to unrestricted bees (p < 0.05, Student’s
t-test, Figure 4), except on days 11 and 16 (p > 0.05).
Mortality during the 16 days experiment was higher (17.7%,
n = 300) in bees deprived of pollen in the first 7 days compared to
bees allowed full pollen supply (7.3%, n = 300, p < 0.05, Mantel-
Cox log-rank test). At the end of the experiment, 16-day-old
adult bees deprived of pollen for the first 7 days had lower fresh
and dry weights of the heads, thorax or abdomen compared to
bees unrestricted in pollen access (n = 60, p < 0.05, Student’s
t-test, Figure 5).
4 Discussion
Larval protein provisioning is important for the development
of the flight apparatus of honey bees (Brodschneider et al., 2009).
Still, the development of flight muscles in the honey bee thorax is
ongoing after adult emergence (Herold, 1965; Hersch et al., 1978;
FIGURE 4
Cumulative pollen consumption of bees with unrestricted access and bees constrained of pollen during the first 7 days. For comparison
between groups at the later stage, consumption of unrestricted bees on days 8–16 is shown seperately. Box and whisker plots (minimum, lower
quartile, median, upper quartile, maximum and outliers) for cumulative consump tion of pollen (mg/bee) by caged honey bees from day 1 until day
16 and from day 8 until day 16, respectively (n = 6 cages of bees for each group). Cumulative lifetime consumption at age 16 days was
significantly different (a, b, c: different letters indicate p < 0.001, one-way ANOVA with Bonferroni multiple comparisons post-hoc test). * indicates
differences in daily consumption after day 8 between bees with constraint con sumption and full consumption (p < 0.05, Student’s t-test). n.s. = not
significant.
FIGURE 5
Fresh and dry weight of head, thorax, and abdomen of bees with
unrestricted access to pollen (day 1–16) and constraint pollen access
(day 8–16). Means and standard deviations are shown: n =60(10bees
out of 6 replicated cages). * indicates p < 0.05, Student’s t-test.
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Brodschneider et al. 10.3389/fphys.2022.1004150
Harrison, 1986; Roberts and Elekonich, 2005; Fernandez-
Winckler and da Cruz-Landim, 2008). Young bees feed on
beebread, and this source of proteins, lipids and other
nutrients is attributed to better health status (Alaux et al.,
2010; DeGrandi-Hofmann et al., 2010), internal gland
development (Hrassnigg and Crailsheim, 1998) and increasing
thorax and dry weight (Hagedorn and Moeller, 1968; Crailsheim
and Stolberg, 1989). So far, no experiment connected adult
protein provisioning and physiological performance of the
flight muscle in honey bees. We closed this gap by
investigating the importance of adult pollen nutrition for
honey bee flight in a flight mill bioassay. We had to develop a
new method of keeping bees in vitro for flight experiments, as
previous trials of flight experiments with caged honey bees failed
(R. Brodschneider, unpublished data). Because healthy bees do
neither defecate in their nest, nor in a cage (see Pavlović et al.,
2022), the ventriculus (midgut) and rectum of caged bees are
densely filled with undigested pollen, water, and feces. The
pressure of the well-stocked intestine on the tracheal system
(including collapsible air sacs) hinders optimal oxygen delivery
and hence flight. To overcome this, we developed the flight
training box used in this experiment (Figure 1). From the age
of 3 days on, bees were every second day allowed to complete
30 min periods of flight training and defecation flights. The
regular defecation in training flights (visible by spots of feces
on the glass walls of the flight box) reduced the fresh weight of
abdomens of caged bees for about 50 mg (compare weights of
caged bees in Figure 3 with weights of caged bees from
experiment 2 not allowed defecation flights in Figure 5,
though those bees were of different ages). The flight trainings
consequently resulted in a high rate of successfully flying bees.
Another validation for emptying the bees’ intestine with the
defecation flights is that the fresh abdominal weight between
pollen-fed and pollen-deprived bees did not differ (Figure 3),
though this was after the extensive flight experiments in which
bees also often defecated.
Adult bees deprived of pollen significantly differ from pollen-
fed bees in several flight characteristics: pollen-deprived bees flew
with lower metabolic power longer and further than pollen-fed
bees, they showed lower flight speed at the beginning of the
flights, (Table 1 ), and they could not raise maximum flight speed
in 2 M glucose fl ights compared to 1 M glucose flights. Bees fly
longer and further when energy-richer 2 M glucose is fed
compared to 1 M glucose solution, but they also increase
speed (von Frisch and Lindauer, 1955; Gmeinbauer and
Crailsheim, 1993; Brodschneider et al., 2009). The flight
curves (Figure 2) show that the first minutes of flights are
characterized by powerful and fast flight, and bees afterwards
steadily reduce speed until carbohydrate reserves in the intestine
and hemolymph are spent (Gmeinbauer and Crailsheim, 1993;
Hrassnigg et al., 2005). We interpret the longer (and thus further)
flights of pollen deprived bees in their lowered ability for high
energy turnover in this period at the beginning of flights. Pollen-
deprived bees are probably impaired in their post-emergence
flight muscle maturation or digestive and other physiological
processes (see Ricigliano et al., 2017) which probably limit
performing full force. Still, all bees received the same amount
of sugar fuel, so pollen-deprived bees spend less fuel per time of
flight and hence flew longer and further in both experimental
flights. The longer flight times with the same energetic
expenditure are also reflected in lower metabolic power
(Nachtigall et al., 1995) in pollen-deprived bees, very similar
to experiments where larval nutrition was manipulated
(Brodschneider et al., 2009).
The flight capacity of honey bees has been proven to be
affected by different biotic and abiotic factors. Some or even all
the factors discussed below could synergistically act together and
drastically impair the flight capacity of bees, which subsequently
reduces collected forage and colony fitness. In this study we
demonstrated the negative impact of pollen malnutrition during
adult bee life, whereas the effect of larval nutrition was shown in
Brodschneider et al. (2009). We can speculate that the worst case
for flight capacity development would be that bees experience
deficient nutrition during both, larval and early adult
development. Pesticides can further impair honey bee flight by
reducing respiration or mitochondrial activity (Hatjina et al.,
2013; Nicodemo et al., 2014; Tosi et al., 2017; Coulon et al., 2020).
In bumble bees, Syromyatnikov et al. (2017) reported that
fungicides used in greenhouses inhibit mitochondria in the
flight muscle. The neonicotinoid pesticide imidacloprid
reduced range and period of bumble bee flights (Kenna et al.,
2019). High, but not field-realistic concentrations of a mito-toxic
fungicide did impair honey bee flight capacity in a flight chamber
(Glass et al., 2021). These authors by the way also reported a
reduced thorax mass of bees reared under field-realistic fungicide
concentrations. Next to the abiotic factors, several honey bee
pests may affect flight physiology. Blanken et al. (2015) showed
that forager bees fly shorter distances when parasitized with the
mite Varroa destructor and exposed to imidacloprid, a
neonicotinoid insecticide, but flight speed remained
unaffected. Another parasitic mite living in the honey bees’
tracheas, Acarapis woodi, reduces the safety margin for
tracheal oxygen delivery during flight in hypoxic air (Harrison
et al., 2001). Dosselli et al. (2016) reported that infection with the
microspiridium Nosema apis affects honey bee workers flight
activity. In contrast to this, Wells et al. (2016) did not confirm
this for bees naturally infected with Nosema ceranae, but
demonstrated that infection with deformed wing virus
reduced flight time and distance.
Some flight characteristics were significantly different
between the two i nvestigated groups, but our results also
show that even adult bees completely deprived of pollen
nutrition can fly. Larval feeding, pupal development and
compromised post-emergence devel opment of the tho rax
obviously suffice for flight, even without adult provision of
pollen derived nutrients. This underlines the importance of
Frontiers in Physiology frontiersin.org08
Brodschneider et al. 10.3389/fphys.2022.1004150
flight musculature for honey bees. We assume a relatively
strong need for adult maturation of the important flight
muscle compared to other organs. In bees, the allocation
tradeoff of nutritional resources in distress , and possible
competitions of different organs or body parts, largely
remain to be expl ored ( Nijhout and Emlen, 1998; Tigreros
and Davidowitz , 2019; Metz and Tarpy, 2022). We need to
emphasize th at in our tether ed flight experiment bees of both
groups were quasi forced to fly, especially at the latter periods
of flights. Slight drawbacks in flight of honey bees in free-
flying environments could be more dramatic, as bees may fail
in returning to the colony after unsuccessful attempts
(Harr ison et al., 2 001). Pollen subst itutes optimized for th e
bees’ needs could partly support their thorax development
when there is insufficient or only nutritionally deficient pollen
available in the environment (Pavlović et al. , 2022; Ricigliano
et al., 2022). In our experiment we established a very drastic
all-or-nothing situation regarding pollen availability of the
two experimental groups. Such long periods of complete
pollen starvation may be rare in a colony with its bee bread
stores, but we can speculate if a sensitive short phase for pollen
feedingexistsinayoungworkerbee’s life, which we
investigated in the second experiment.
In this feeding experiment we tested if bees perceive a
complete protein deficit experienced during the fi rst days after
emergence and compensate for, or at least try to compensate
for. Such a compensation could be manifested in an increased
consumption at a later stage, to levels higher than measured in
bees of the same age, but never restric ted in pollen. A cert ain
flexibility in age-related behavior or gland development is
common in honey bees (Cr ailsheim und S tolberg, 1989;
Robinson, 1992; Hrassnigg and Crailsheim, 1998; Schmickl
and Crailsheim, 2004). Indeed, bee s pre vented from feeding
onpolleninthefirst 7 days of their life s ubsequently fed more
pollen on almost every day at ages 8–16 days, compared to
bees that always had full access to pollen (Figure 4). This
evidences that the bees perceive the pollen deficit and increase
consumption to compensate for the early life deprivation.
Nonetheless, the cumulative lifetime pollen intake o f bees
deprived of pollen the first 7 days resulted at the end of the
experiment on day 16 in only about half (51.1%) of the amount
of pollen ingested compared to bees unrest ricted in pollen. If
the experiment would have lasted a few days longer, the
lifetime consumption would have only increased
marginally, as can be seen by the fl
attening of the
cumulative c onsumpt ion c urve (Figure 4). As expected,
caged bees h aving full access to pollen consumed most of
the pollen at the first 5 days of their life (Dietz, 1969;
Crailsheim et al., 1992 ; Omar et al., 2017). The reduced
lifetime pollen consumption of constrain t bees suggests t hat
older bees’ physiology does not allow them to catch up with
the protein consumption missed during early life. Reasons for
this could be the transit ion in ab ility of older b ees to dige st
pollen diets, the atrophy of hypopharyngeal glands or the
absence of protein in the bees’ early diet which could influence
digestion or metabolism (Moritz and Crails heim, 1987;
Crailsheim and Stolberg, 1989; Ricigliano et al., 2017).
Because of the critical phase for adult pollen nutrition
identified in our study, some age cohorts in colonie s may
already suffer from shorter periods of pollen dearth than
previously thought. For these distressed bees, even a good
pollen forage after a severe shortage is not a solid remedy of
the malnourishment (Requier et al., 2017).
In both experiments, full and early-life restrictions to
protein-free diets resulted in reduced body weight of worker
bees. This was particularly the case for the fresh and dry weight of
the thorax, site of the flight muscles. In the second experiment we
constraint bees of pollen only for the first 7 days of their life,
when bees usually consume pollen (Dietz, 1969; Moritz and
Crailsheim, 1987; Crailsheim et al., 1992). Deprivation at this
early age is sufficient to significantly reduce thorax weight. A
delayed availability of protein food for bees older than 7 days did
not allow full thorax weight compensation of the deficits
experienced during early life, whereas starvation during only
the first 3 days of life could be compensated later (Crailsheim and
Stolberg, 1989). Aside from the thorax, also head weight is
reduced, which is an indication for impaired hypopharyngeal
gland development (Hrassnigg and Crailsheim, 1998). Bees
feeding on pollen later in life than usual experience early
adult life stress (malnutrition in a sensitive feeding period)
which affects body weight and probably life history.
Accordingly, we found that early adult life pollen deprivation
for 7 days is enough to stunt honey bee survival in vitro, whereas
significant impacts on longevity so far have mostly been
established for life-long pollen deprivation (Omar et al., 2017;
Khedidji et al., 2022) or when bees were offered only a lowered
share (40%) of the amount of oilseed rape pollen usually
consumed (di Pasquale et al., 2016). Consequences of early
life stress in honey bees have so far been mostly investigated
by manipulating parasite or nutrition levels during the
developmental stages (Sco
field and Mattila, 2015; Rueppel
et al., 2017). Amdam et al. (2009) on the contrary studied
brood-rearing levels of young adult bees and found these to
be linked to life trajectories. They found that no brood-rearing
activity of young bees is associated with high vitellogenin protein
levels, a later onset of foraging and longer life expectancy. In our
experiment bees experienced nutritional stress in their early adult
life which caused lower body weight and longevity. Such bees
may be an interesting new study subject for scientific research of
early life deprivation or as an intermediate group between fully
fed and unfed bees.
Our fi ndings underline the importance of nutr ients from
pollen for the development of adult honey bees. We provide
evidence for the need of adult pollen n utrition in terms of
flight muscle mass, and maximal flight force. We further
detected a sensitive phase of pollen feeding in the first
Frontiers in Physiology frontiersin.org09
Brodschneider et al. 10.3389/fphys.2022.1004150
7 days of adult life of honey bees. Bees deprived of pollen
during this critical p eriod for protein consumption can
perceive this shortcoming and try to compensate it by
increasing their daily pollen consumption later in life.
Unfortunately, their a ge-relat ed physiological constitution
does not allow them to consume enough pollen to reach a
full lifetime amount of protein. The deficiencies acquired
during this early adult-life deprivation cannot be f ully
compensated as w e showed for body mass and longevity
and possibly affect honey bee life trajectories and health.
Data availability statement
The raw data supporting the conclusion of this article will be
made available by the authors, without undue reservation.
Author contributions
Conceptualization: RB and KC; Formal analysis: RB and EO;
Investigation: EO and RB; Writing—original draft preparation:
RB, EO, and KC; Supervision: KC.
Funding
EO received a grant from Egypt’sOffice for Cultural and
Educational Relations, Embassy of the Arab Republic of
Egypt.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily r epresent those of their
affiliated organizations, or those of the publisher, the
editors and the reviewers. Any product that may be
evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the
publisher.
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