The use of a within-hive replication bioassay method to
investigate the phagostimulatory effects of pollen, bee
bread and pollen extracts, on free-flying honey bee
colonies
Richard James Bridgett, William Daniel John Kirk, Falko Pieter Drijfhout
To cite this version:
Richard James Bridgett, William Daniel John Kirk, Falko Pieter Drijfhout. The use of a
within-hive replication bioassay method to investigate the phagostimulatory effects of pollen,
bee bread and pollen extracts, on free-flying honey bee colonies. Apidologie, Springer Verlag,
2015, 46 (3), pp.315-325. .
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Apidologie (2015) 46:315–325
* INRA, DIB and Springer-Verlag France, 2014
DOI: 10.1007/s13592-014-0324-z
Original article
The use of a within-hive replication bioassay method to
investigate the phagostimulatory effects of pollen, bee bread
and pollen extracts, on free-flying honey bee colonies
Richard James BRIDGETT1 , William Daniel John KIRK2 , Falko Pieter DRIJFHOUT1
1
Chemical Ecology Group, School of Physical and Geographical Sciences, Keele University, Staffordshire, UK
2
School of Life Sciences, Keele University, Staffordshire, UK
Received 24 June 2014 – Revised 21 August 2014 – Accepted 29 September 2014
Abstract – A method for conducting multiple-choice bioassays, incorporating within-hive replication, is described
and demonstrated here. This method has been used to study the influence of phagostimulants in pollens on food
uptake within honey bee colonies. Experiments using bee-collected trapped pollens and a sample of stored bee bread
suggest that there is little or no difference in the preference of bees towards fresh pollen or bee bread. Further work
using solvent extracts of pollen showed that phagostimulants are easily extractable in sufficient quantities to increase
the consumption of artificial diet in field-based colonies, despite alternative natural forage being available. Data
indicate that the addition of polar solvent extracts of pollen increases diet consumption more than less polar extracts.
Isolating and identifying phagostimulants could contribute towards production of a range of more palatable artificial
diets than those currently available.
Apis mellifera / phagostimulant / diet / feeding / forage
1. INTRODUCTION
Numbers of honey bee colonies (Apis
mellifera ) have fallen sharply over recent years
across Europe and North America (Van
Engelsdorp et al. 2008, 2010; Aston 2010;
Carreck and Neumann 2010; Potts et al. 2010;
van der Zee et al. 2012). One way in which
beekeepers may attempt to stimulate colony population growth is to offer high-protein artificial or
supplemental diets to colonies. Commercial diets
are available, but at times, these can be poorly
consumed by bees compared to pollen (DeGrandiHoffman et al. 2010; Saffari et al. 2010a, b).
Although bees generally prefer pollen to a pollen
substitute, the addition of small amounts of
Corresponding author: R. Bridgett,
[email protected]
Manuscript editor: Peter Rosenkranz
natural pollen improves the uptake of artificial or
supplemental diets within the hive, thereby contributing to better overall colony health during
times of sparse pollen (Herbert et al. 1980;
Standifer 1980; Manning et al. 2007). However,
adding pollen to diets has risks associated with it,
such as transmission of pathogens between colonies or the addition of pollens contaminated with
toxic substances (Herbert et al. 1980; Schmidt
et al. 1987; Saffari et al. 2010b). Such risks can
be minimised by subjecting pollen to a prefeeding sterilisation process, such as the irradiation or heat treatments described by Williams et al.
(2013), although this is time-consuming and may
be unwanted expense for beekeepers. A recently
developed commercial diet has been reported to
be as palatable as pollen to bees (Saffari et al.
2010a, b). Unfortunately, the reason for the increased acceptability of this diet has not been
identified, and diet palatability here was assessed
partly using no-choice feeding trials. More confidence could be placed in this result if
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R.J. Bridgett et al.
colonies were offered artificial diet and pollen in
choice trials; further research into increasing the
palatability of artificial diets is required.
Pollen palatability is not solely linked to the
protein content of pollens (Schmidt 1984).
Sunflower (Helianthus annuus ) pollen has a protein content of only 15 % (Pernal and Currie
2000), and studies into colony longevity have
shown that although colonies can perform poorly
when fed this nutritionally deficient pollen alone,
bees find sunflower pollen highly palatable
(Schmidt et al. 1995). Pollens also contain extractable naturally occurring feeding stimulants
(phagostimulants) (Doull 1973; Schmidt 1985;
Schmidt et al. 1987; Dobson and Bergstrom
2000; Schmidt and Hanna 2006). Previous work
has suggested that the lipid-based adhesive substance pollenkitt (in addition to other functions)
may be responsible for making pollen attractive to
pollinators (Pacini and Hesse 2005), whilst another study shows that a particular long-chain fatty
acid (trans , cis , cis ,-2,9,12 octadecatrienoic acid)
acts as a strong attractant towards foraging bees
(Hopkins et al. 1969). Unfortunately, no other
examples of studies confirming the action of this
fatty acid have been found, and it may be unwise
to perpetuate assertions from historical studies
that have not been reconfirmed. Therefore, despite
these assertions, the chemical nature of
phagostimulants has more recently been reported
as remaining unknown (Schmidt and Hanna
2006).
Isolating and identifying additional
phagostimulants could contribute towards the production of a range of more palatable artificial
diets. Using these could mitigate against the risks
associated with adding natural pollen to diets,
thereby allowing beekeepers to strengthen their
colonies more safely. To achieve this, more understanding of their chemical nature and origin is
required.
Foraging bees do not consume the pollen that
they return to the hive. It is off-loaded to other
colony members that facilitate its processing.
Foraged pollen is packed into comb cells, covered
with honey, and undergoes a chemical conversion
into bee bread prior to consumption (Vasquez and
Olofsson 2009). The conversion processes include bacterial lactic acid fermentation but are
not fully understood (Gilliam et al. 1989;
Vasquez and Olofsson 2009). The resultant bee
bread has a lower pH than pollen, helping to
prevent detrimental microbial growth and spoilage (Loper et al. 1980; Herbert and Shimanuki
1978). Bee bread may also be of greater nutritional value, as lactic acid bacteria could enhance the
availability of vitamins B7, B11 and B12, vitamin
B12 not being naturally available from plant products (Vasquez and Olofsson 2009). Additionally,
it is partially pre-digested, with some authors
speculating that the end product may be more
palatable to bees than fresh pollen (Loper et al.
1980). The presence of compounds identified as
attractants to foraging bees in pollens (Hopkins
et al. 1969) does not necessarily mean that the
same compounds function as phagostimulants for
consumption within the hive. There are documented examples of foraged pollens being poorly
consumed once returned to the hive and, of certain
pollens, being consumed despite exhibiting some
level of toxicity towards bees (Schmidt et al.
1995).
Previous phagostimulant studies have shown
that increased consumption of artificial diets can
be achieved if solvent extracts of pollens are
added. Studies indicate that both polar and nonpolar solvent extracts of pollen can provide increased stimulation (Robinson and Nation 1968;
Doull and Standifer 1970; Doull 1974b; Schmidt
1985; Schmidt and Hanna 2006). What is not
clear, however, is whether compounds extracted
by polar or non-polar solvents increase diet consumption more than the other or, given that bees
do not consume freshly foraged pollen (Herbert
and Shimanuki 1978), whether there may be a
more suitable pollen material to use than freshly
trapped pollen (corbicular pollen pellets) in
attempting to isolate phagostimulants. If bee bread
is more palatable than fresh pollen, this may indicate that additional phagostimulants are present
following the conversion process. If so, using
bee bread in studies aimed at identifying
phagostimulants would be more appropriate.
The relative palatability of different diets to
colonies can be assessed more rigorously by offering multiple diets to colonies, alongside control
diets. It would be of additional benefit if methods
existed that enabled the gathering of sufficient
Phagostimulatory effects of pollen
data to perform appropriate statistical analysis,
without the need for large numbers of experimental colonies. Being able to perform within-hive
replication would allow enough replicates to perform statistical analyses, but with a decreased
number of colonies. Six experiments using freeflying field colonies are communicated here.
Experiments 1–3 focussed on ensuring that the
method was suitable to use in conducting
multiple-choice bioassays incorporating withinhive replication. Experiments 4–6 used this bioassay method to test the following: (1) whether or
not there was a significant difference between the
phagostimulatory effects of a sample of bee bread
and bee-collected trapped pollen (experiments 4a
and 4b), (2) whether extractable phagostimulants
from pollens could increase the uptake of artificial
diet in the presence of alternative natural forage
(experiment 5) and (3) whether polar or non-polar
solvent extracts of pollens provide greater feeding
stimulation to pollen-consuming bees (experiment
6).
2. MATERIALS AND METHODS
Honey bee (A. mellifera ) colonies used within these
studies were free-flying and located in a semi-rural
environment between open farmland and urban development within the UK. They were housed in modified
46×46-cm British national hives. These consisted of
one brood box and either one or two supers, each
holding ten frames. Colonies were of approximately
equal strength, with bees on at least eight frames within
the brood chamber. Freshly foraged pollen was trapped
using a hive entrance pollen trap throughout 2012 and
2013 and stored at −40 °C until use.
All bioassays utilised an artificial diet consisting of
(by mass) 65 % commercially available soya flour
(NBTY Europe Ltd), 20 % commercially available
vegetable fat (TREX, Edible oils Ltd) and 15 % icing
sugar (beet sugar, Silver Spoon, UK) as either a control
or a substrate diet to which additions could be added for
activity testing as treatments. Small amounts of cool tap
water were added to diets to facilitate their binding into
a dough-like texture. This formulation is an adaption of
a publicised recipe by the Australian Government’s
Rural Industries Research and Development
Corporation (Somerville 2005). Diets were prepared as
a single bulk diet before being split and administered to
317
colonies in patty form within small 7 mm deep, 3.5 cm
by 3.5 cm, weighing boats (Fisher Scientific, UK). Patty
surface area can influence consumption, with larger
surface area patties being consumed more quickly
(Avni et al. 2009). Using weighing boats to deliver
patties in allowed standardisation of individual patty
surface area to 12.25 cm2 each, therefore eliminating
this as a potential problem. Colonies were double queen
excluded (Herzog type) with patties placed, upturned, in
the bee space created between the queen excluders,
above the brood nest. Patties were positioned regularly
in a grid pattern to ensure even distribution of treatments
across the colony, therefore minimising any positional
bias. Patties were placed approximately 4 cm from each
other; with no patty positioned above an area of the
brood chamber not populated by bees. Diet consumption was measured by monitoring mass loss over time,
with bee preference inferred from this. Trials were conducted over a period of 4 days, unless complete consumption of a patty was observed, at which point trials
were halted so as not to let the change in available diets
(alteration of choice) affect the outcome.
2.1. Bioassay method development
Performing within-hive replication leads to increased variation in the consumption of any given diet,
due to spatial variation in the placement of patties
relative to the bulk of the brood nest. To test whether
the bioassay method is appropriate and can effectively
distinguish between diets of differing palatability, despite this spatial variance, two experiments were
conducted.
2.1.1. Experiment 1—bioassay of identical
palatable diets only
To ensure that spatial variation in patty placement
and consumption does not produce a false positive
result, four identical palatable diets were offered to
colonies. These were formulated by replacing 10 %
(by mass) of the described diet formulation with trapped
pollen; 55 % of the whole remained as soya flour, with
the amounts of vegetable fat and sugar unchanged at 20
and 15 %, respectively. Trapped pollen pellets were
lightly broken down using a pestle and mortar to separate grains prior to addition to the diet. This aided
mixing and enhanced distribution throughout the diets.
The four diets were arbitrarily designated replicate A, B,
318
R.J. Bridgett et al.
C and D, respectively. Each was split into 16 individual
patties weighing 9.5±0.5 g each and offered to four
colonies. Four patties of each diet were administered
to each colony in a regular arrangement (16 patties in
total), and their mass loss over 4 days was recorded.
Data were then statistically analysed to test whether
there was any significant difference in the consumption
of the four identical diets.
2.1.2. Experiment 2—bioassay of control
versus treatment diets
To ensure that increased spatial variation does not
prevent the detection of a difference in diet palatability
overall, a bioassay with two control diets (without pollen) of the formulation described at the beginning of this
section (controls A and B) and two treatment diets
(treatments C and D) was performed. Treatment diets
were prepared as described for experiment 1, incorporating 10 % trapped pollen. These four diets were then
offered to colonies in the same manner as the previous
experiment, with two control (eight patties) and two
treatment diets (eight patties) per colony. Consumption
data were statistically analysed to confirm that the two
diet types could be distinguished from each other.
2.1.3. Experiment 3—passive moisture loss
simulation
A third experiment aimed at approximating the
greatest degree of mass loss due to the passive drying
out, (moisture loss) of diets in colonies over the test
period was performed. A diet of control formulation
was prepared as before. This was split into 32 individual
patties of 9.5±0.5 g that were placed within an incubator. The incubator was kept at 34 °C to simulate colony
temperature, and mass loss of the individual patties was
recorded at 24-h intervals over a period of 10 days.
2.2. Palatability testing
2.2.1. Experiments 4a and 4b—the
phagostimulatory effects of bee bread and
fresh pollen
To assess the relative palatability of fresh pollen and
bee bread, two identical bioassays (experiments 4a and
4b) were performed consecutively. Within these, treatment diets containing 10 % bee bread and 10 % fresh
pollen and a control diet were offered to colonies. This
comparison requires that both pollen forms contain
broadly the same pollen species. Attempts to perform
this comparison may be confounded if this is not the
case. For this experiment, trapped pollen was collected
on 8 May and 24 May, 2012, and bee bread was removed from a frame on 24 May 2012. Pollen and bee
bread were stored at −40 °C until use.
Samples of the most common colour observed by
eye from both the trapped pollen and bee bread samples
were mounted and examined microscopically according
to the method described by Sawyer (1981). Pollens
were identified with the aid of a reference set of
mounted pollen grains within the School of Life
Sciences at Keele University. Under natural conditions,
it is almost impossible to ensure that bee bread samples
contain identical pollens to trapped samples, but sampling of both was done in the spring, when bee bread in
colonies from the previous season would likely have
been used up, to maximise the chance of pollen overlap.
Diets were prepared as before and split into individual patties weighing 8.5±0.5 g each at placement. Four
colonies were used within these experiments, with 16
replicates of each diet (four control, four trapped pollen
treatment and four bee bread treatment per colony) used
in each experiment.
2.2.2. Experiment 5—phagostimulant effect
on free-flying colonies
This experiment assessed whether the addition of
solvent extracts containing phagostimulants could increase the consumption of artificial diet in free-flying
field colonies, in the presence of alternative natural
forage. A series of ultrasonic-assisted solvent extractions, within an increasing polarity gradient, using n hexane, ethyl acetate and methanol (HPLC-grade solvents, Fisher Scientific), were performed on samples of
mixed-species trapped pollens. Extractions were performed in series on the same pollen sample. Extraction
with n -hexane was followed by extraction with ethyl
acetate and then methanol. These solvents were used to
ensure that compounds of differing polarities were all
efficiently extracted from the pollens.
Seven millilitres of extraction solvent were added
per 1 g of pollen to be extracted within a 50-mL centrifuge tube. This was then vortexed for 1 min before
being placed in an ultrasonic bath for a further 30 min.
The temperature of the water bath was not allowed to
Phagostimulatory effects of pollen
exceed 45 °C. Following this, extracts were centrifuged
as required to remove pollen solid from the solvent,
before the supernatants were removed for further use.
Extracts were pooled, and the solvent was evaporated as
far as possible. The resultant slurry was then added to a
diet of the formulation described previously to give a
treatment diet containing pollen extract.
The remaining pollen, post-extraction, was then
added to another diet, forming a second (extracted pollen) treatment diet. A third diet of the previous formulation alone provided a control to test against these
treatments. Treatment diet patties were prepared before
being left for 48 h to facilitate complete solvent evaporation. The control diet, the treatment diet containing the
solvent extract and the treatment diet containing the
post-extraction pollen were then offered to colonies.
Three colonies were used, with five replicates of each
of these diets placed in each. Individual patties weighed
8.5±0.5 g at placement. The experiment was halted
after 3 days.
2.2.3. Experiment 6—the effect of solvent
extract polarity on artificial diet consumption
To compare the effect that solvent extracts (varying
in polarity) of pollens have on diet consumption, another series of ultrasonic-assisted solvent extractions were
performed on two samples of mixed-species trapped
pollens. The described extraction procedure of experiment 5 was repeated. As well as ensuring efficient
compound extraction, it is expected that utilising these
solvents also facilitates some crude segregation (based
on polarity) of extracted compounds between the extracts. Again, extractions were performed in series, with
all three solvent extractions performed on both pollen
samples. The solvents were evaporated almost to dryness, leaving two n -hexane, two ethyl acetate and two
methanol extracts to be added to diets as required.
One n -hexane, one ethyl acetate and one methanol
extract were pooled and added to an artificial diet of the
formulation given previously to use as a control. In the
same way, three additional treatment diets were formulated, but each treatment diet only had one solvent
extract (n- hexane, or ethyl acetate, or methanol)
added. All four diets were split into patties before again
being left for 48 h to facilitate complete solvent evaporation. They were again distributed across the four
brood chambers in a regular manner, with 16 replicates
(four per colony) of each of the diets offered in total.
319
Patties weighed 8.5±0.5 g at placement. This experiment was halted after day 3.
2.3. Statistical analysis
Previous studies have highlighted that feeding bees
will not consume bee bread which is located more than
7 cm from the brood; stores located further than this are
discovered only by chance (Doull 1974a; van der Steen
2007). Each patty is considered an independent observation within this method. Data were log10 (x ) transformed to homogenise the variance and normalise the
distribution, before being analysed by ANOVA within
Minitab 16 (Minitab Inc., USA). ANOVAs of experiments 1, 2, 3, 5 and 6 were conducted with colony
considered as a block. The results of experiments 4a
and 4b, carried out to assess the relative phagostimulatory effects of pollen and bee bread, were combined
after confirming the absence of a treatment × experiment interaction. This allowed statistical analysis to be
conducted with 32 replicates, and experiments 4a and
4b are hereafter referred to as experiment 4. In experiment 4, colony and experiment were considered as
blocks to remove the between-colony and betweenexperiment variation. The regular distribution of different diets minimises the effect of spatial variation in
consumption from the density of brood below. See
Figure 1 for an illustration of a typical arrangement of
Figure 1. Diagram showing an example of regular patty
placement across a brood chamber. Different letters
correspond to different replicate diets used within experiment 1. Individual patties were positioned 4 cm
away from neighbouring patties.
320
R.J. Bridgett et al.
patties across a brood chamber. Multiple comparisons
of individual treatments used Tukey’s test.
3. RESULTS
3.1. Experiment 1—bioassay of identical
palatable diets only
There was no significant effect of diet (F 3,57 =
0.00, P =1.00) (Figure 2), confirming that the
method was not producing false positives.
3.2. Experiment 2—bioassay of control
versus treatment diets
Data from a colony that swarmed were excluded from the analysis, leaving 12 replicates of each
diet type. There was a significant effect of diet on
c o n s u m p t i o n (F 3 , 4 2 = 1 3 5 . 3 6 P < 0 . 0 0 1 )
(Figure 3). There was a significant difference
Figure 3. Back-transformed mean (±SEM) consumption (g per patty) of the two control and two palatable
10 % pollen diets. Trial duration was 4 days, and 12
replicate patties of each diet were used. Consumption of
diets that share the same letter did not significantly
differ (P <0.05).
between the mean consumption of control A compared to treatment C and treatment D (both
P <0.001), but there was no significant difference
between the consumption of control A and control
B (P =0.99). Likewise, there was a significant
difference between the mean consumption of control B and treatment C and treatment D (both
<0.001). There was no difference in the mean
consumption of treatment C and treatment D
(P =1.00).
3.3. Experiment 3—passive moisture loss
simulation
Figure 2. Back-transformed mean (±SEM) consumption (g per patty) of four identical palatable 10 % pollen
treatment diets. Trial duration was 4 days, and 16 replicate patties of each diet were used. Consumption of
diets that share the same letter did not significantly
differ (P <0.05).
The mean cumulative mass loss of diets over a
10-day period is shown in Figure 4. The mean
decrease in mass due to passive moisture loss
alone was 0.855±0.01 and 0.971±0.01 g after 3
and 4 days, respectively. This equates to 10 % of
the mean initial mass.
Phagostimulatory effects of pollen
321
Figure 4. Cumulative mean mass loss (±SEM) per control patty over the 10-day period within the incubator
(n =32).
There was a significant difference between the
mean consumption of control diet and trapped
pollen diet (P <0.001) and control diet and bee
bread diet (P <0.001). However, there was no
significant difference between the consumption
of trapped pollen diet and bee bread diet (P =
0.12).
The trapped pollen samples contained predominantly pollens from species within the Rosaceae,
with lesser amounts from the Asteraceae and
Liliaceae. There was good overlap with the bee
bread samples, with the most abundant grains
observed here also Rosaceae. However, there
were fewer unidentified pollens in the bee bread
samples. At the time of collection, species
flowering within foraging distance of the colonies
included Rosaceae (hawthorn, cherry, apple, pear,
rowan), Asteraceae (dandelion) and Liliaceae
(bluebell).
3.4. Experiment 4—the phagostimulatory
effects of bee bread and fresh pollen
3.5. Experiment 5—phagostimulant effect
on free-flying colonies
There was a significant effect of diet on consumption (F 2,89 =116.36, P <0.001) (Figure 5).
There was again a significant effect of diet on
consumption (F 2,40 =28.33, P <0.001) (Figure 6).
There was a significant difference between the
Figure 5. Back-transformed mean (±SEM) consumption (g per patty) of the control, 10 % pollen and 10 %
bee bread diets. Trial duration was 4 days, and 32
replicate patties of each diet were used. Consumption
of diets that share the same letter did not significantly
differ (P <0.05).
Figure 6. Back-transformed mean (±SEM) consumption (g per patty) of the control, pollen extract and
extracted pollen diets. Trial duration was 3 days, and
15 replicate patties of each diet were used. Consumption
of diets that share the same letter did not significantly
differ (P <0.05).
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R.J. Bridgett et al.
consumption of all three test diets: control and
pollen extract diet (P <0.001), control and extracted pollen treatment (P <0.001), and pollen extract
treatment and extracted pollen diet (P =0.01).
3.6. Experiment 6—the influence of varying
solvent polarity pollen extracts on artificial diet
consumption
There was a significant effect of diet on consumption (F 3,56 =4.71, P =0.01) (Figure 7). There
was a significant difference between the mean
consumption of the pooled extract control diet
and the diet containing hexane extract alone
(P =0.01). There was no significant difference in
the mean consumption of the control diet and the
diet containing ethyl acetate extract only (P =
0.07) or the diet containing methanol extract only
(P =0.89). There was also no significant difference in the mean consumption of the diet containing the hexane extract only and the diet containing
the ethyl acetate extract only (P =0.85) or the diet
containing the methanol extract only (P =0.06).
Figure 7. Back-transformed mean (±SEM) consumption (g per patty) of the combined pollen extract (control), hexane extract, ethyl acetate and methanol extract
diets. Trial duration was 3 days, and 16 replicate patties
of each diet were used. Consumption of diets that share
the same letter did not significantly differ (P <0.05).
There was no significant difference in the consumption of the diet containing ethyl acetate extract only and the diet containing methanol extract
only (P =0.30).
4. DISCUSSION
Experiments 1 and 2 showed that regular distribution of treatments above the brood within
each hive adequately reduced any bias from spatial variation of the brood below. The method did
not produce false positives, and it correctly distinguished between palatable and less palatable diets.
Data from experiment 3 suggest that after 3 or
4 days, mass loss of up to approximately 1 g
(10 %) may be expected, due to passive moisture
loss alone. This data should be treated with a
degree of caution though, as although approximate colony temperature could be replicated,
there was no facility to replicate colony humidity.
Increased colony humidity is almost certainly going to influence the rate of moisture loss of patties
placed within. However, it is unlikely that it
would increase the rate of moisture loss above
that observed here, and mass loss of greater than
10 % of patties is therefore likely due to consumption by bees.
The experiment assessing the phagostimulatory effects of bee bread and fresh pollen (experiment 4) showed that bees exhibited no significant
preference for diets containing either freshly foraged pollen or a sample of bee bread. In this
instance, these results do not support previous
suggestions that bees may find bee bread more
palatable (Loper et al. 1980). The addition of 10 %
by mass of pollen or bee bread significantly increased diet uptake. As there was no indication of
an increase in phagostimulant abundance due to
the pollen conversion process, it seems acceptable
to use freshly trapped pollen pellets in work aimed
at identifying phagostimulants; trapped pollen is
easier to gather and manipulate than bee bread,
and no preference towards bee bread was observed here.
The lack of a preference for either diet may also
reveal additional information relating to the chemical nature of phagostimulants in pollens.
Considering the function of phagostimulants
may be important when devising identification
Phagostimulatory effects of pollen
methods. Volatiles are easily analysed using gas
chromatography, but less volatile, more polar,
compounds are likely to require the use of alternative analytical methods in their identification. If
phagostimulants are volatile, functioning via olfactory stimulation, it might be expected that their
presence in bee bread would be diminished due to
time spent within the conversion process and
evaporation at colony temperature. This would
likely result in a significant difference between
the consumption levels of trapped pollen and bee
bread. These data suggest that consumption was
broadly similar, possibly indicating that
phagostimulants are more likely detected in the
taste of a food source (functioning as a gustatory
stimulant) once bees have commenced feeding.
This hypothesis is perhaps also supported by data
highlighting that extremely non-polar compounds
(therefore likely to include volatiles) in certain
pollen extracts seem to offer no phagostimulatory
effect (Schmidt 1985).
The experiment assessing the strength of extractable phagostimulants on free-flying colonies
(experiment 5) indicates that stimulants are easily
extractable from pollens in sufficient quantities to
increase uptake of an artificial diet, even in the
presence of alternative natural forage during summer. The consumption of the pollen extract diet
was approximately five times greater than the
control, while the extracted pollen diet was consumed significantly more than the control, but less
than the pollen extract diet. This indicates that
complete extraction of the phagostimulants in this
manner is not achieved. The increased consumption of the diet containing extracted pollen over
the control diet reveals either that some
phagostimulants are not extractable from pollen
via this method or, more likely, that increased
volumes of solvent would be needed to achieve
100 % extraction.
The control diet within the final experiment
consisted of all three solvent extracts combined.
In this instance, the phagostimulatory effects of
the less polar hexane extract were significantly
less than all three solvent extracts combined.
However, neither the ethyl acetate nor methanol
extract individually provided significantly less
stimulation than the positive control. The observed mean consumption values themselves
323
may suggest a trend in the phagostimulatory activity of extracts of n -hexane extract < ethyl acetate extract < methanol extract < combined extracts. No other examples of multiple-choice feeding trials directly comparing the activity of different polarity solvent extracts have been found, but
these data appear to indicate that the more active
phagostimulants reside in more polar solvent extracts. While these data support previous data in
suggesting that the greatest stimulation may result
from the summative effects of polar and non-polar
compounds (Schmidt and Hanna 2006), potentially there are specific, more polar compounds that
can provide significant stimulation themselves.
Future research into the phagostimulatory activity
provided by fractions of polar extracts would be
highly beneficial. However, achieving isolation
and identification of polar compounds acting as
phagostimulants may present significant challenges. These supplemental feeding trials join a
substantial list of previous nutritional studies,
summarised by Brodschneider and Crailsheim
(2010). The consumption rates (g/day) of the supplemental diets within these most recent studies
vary but are comparable to some of those previously observed. The colonies used within these
studies were free-flying field colonies, with no
restrictions placed upon them. Although it is perhaps preferable to conduct such experiments using
large numbers of colonies, this within-hive replication method offers a viable alternative, should
financial or infrastructural constraints prevent the
use of more colonies.
ACKNOWLEDGMENTS
This research was supported by the Perry Foundation and the British Beekeepers Association (BBKA).
We thank David Buckley for the loan of his colonies
and practical beekeeping support, and David Aston and
Pam Hunter of the BBKATechnical Committee for their
specialist advice.
Méthode par essai biologique avec réplications à
l'intérieur de la ruche, pour examiner les effets
phagostimulants du pollen, du pain d'abeille et des
extraits de pollen sur des colonies d'abeilles en vol libre
324
R.J. Bridgett et al.
Apis mellifera / régime alimentaire / nourrissage /
approvisionnement
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