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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

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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:

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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 pop-ulation 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 (DeGrandi-Hoffman et al. 2010; Saffari et al. 2010a, b). Although bees generally prefer pollen to a pollen substitute, the addition of small amounts of

natural pollen improves the uptake of artificial or supplemental diets within the hive, thereby con-tributing 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 colo-nies 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 pre-feeding sterilisation process, such as the irradia-tion 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 in-creased acceptability of this diet has not been identified, and diet palatability here was assessed partly using no-choice feeding trials. More confi-dence could be placed in this result if

Corresponding author: R. Bridgett, r.j.bridgett@keele.ac.uk

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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 pro-tein 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 extract-able 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 sub-stance pollenkitt (in addition to other functions) may be responsible for making pollen attractive to pollinators (Pacini and Hesse2005), whilst anoth-er 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).

I s o l a t i n g a n d i d e n t i f y i n g a d d i t i o n a l phagostimulants could contribute towards the pro-duction 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 un-derstanding 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 in-clude 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 spoil-age (Loper et al. 1980; Herbert and Shimanuki 1978). Bee bread may also be of greater nutrition-al vnutrition-alue, as lactic acid bacteria could enhance the availability of vitamins B7, B11and B12, vitamin

B12not being naturally available from plant

prod-ucts (Vasquez and Olofsson2009). 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 docu-mented 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 non-polar solvent extracts of pollen can provide in-creased stimulation (Robinson and Nation1968; Doull and Standifer1970; Doull1974b; Schmidt 1985; Schmidt and Hanna 2006). What is not clear, however, is whether compounds extracted by polar or non-polar solvents increase diet con-sumption 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 indi-cate that additional phagostimulants are present following the conversion process. If so, using bee bread in studies aimed at identifying phagostimulants would be more appropriate.

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data to perform appropriate statistical analysis, without the need for large numbers of experimen-tal colonies. Being able to perform within-hive replication would allow enough replicates to per-form statistical analyses, but with a decreased number of colonies. Six experiments using free-flying field colonies are communicated here. Experiments 1–3 focussed on ensuring that the method was suitable to use in conducting multiple-choice bioassays incorporating within-hive replication. Experiments 4–6 used this bio-assay 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 devel-opment 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 (Somerville2005). Diets were prepared as a single bulk diet before being split and administered to

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 cm2each, 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 consump-tion was measured by monitoring mass loss over time, with bee preference inferred from this. Trials were ducted over a period of 4 days, unless complete con-sumption 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 withhive replication leads to in-creased 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, de-spite this spatial variance, two experiments were conducted.

2.1.1. Experiment 1—bioassay of identical palatable diets only

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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 pol-len) 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, incorpo-rating 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 incuba-tor. 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 . E x p e r i m e n t s 4 a a n d 4 b—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, treat-ment 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 re-moved 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 sam-pling 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 individ-ual 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 in-crease 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 sol-vents, Fisher Scientific), were performed on samples of mixed-species trapped pollens. Extractions were per-formed 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.

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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 pol-len) treatment diet. A third diet of the previous formu-lation alone provided a control to test against these treatments. Treatment diet patties were prepared before being left for 48 h to facilitate complete solvent evapo-ration. 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, anoth-er sanoth-eries of ultrasonic-assisted solvent extractions wanoth-ere performed on two samples of mixed-species trapped pollens. The described extraction procedure of experi-ment 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 ex-tracts. Again, extractions were performed in series, with all three solvent extractions performed on both pollen samples. The solvents were evaporated almost to dry-ness, 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 formu-lated, 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 evapo-ration. 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.

Patties weighed 8.5±0.5 g at placement. This experi-ment 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 (Doull1974a; van der Steen 2007). Each patty is considered an independent obser-vation within this method. Data were log10(x ) trans-formed to homogenise the variance and normalise the distribution, before being analysed by ANOVA within Minitab 16 (Minitab Inc., USA). ANOVAs of experi-ments 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 phagostimula-tory effects of pollen and bee bread, were combined after confirming the absence of a treatment × experi-ment interaction. This allowed statistical analysis to be conducted with 32 replicates, and experiments 4a and 4b are hereafter referred to as experiment 4. In experi-ment 4, colony and experiexperi-ment were considered as blocks to remove the colony and between-experiment variation. The regular distribution of differ-ent diets minimises the effect of spatial variation in consumption from the density of brood below. See Figure1for an illustration of a typical arrangement of

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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 (F3,57=

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 exclud-ed 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 ( F3 , 4 2= 1 3 5 . 3 6 P < 0.001)

(Figure 3). There was a significant difference

between the mean consumption of control A com-pared 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 con-trol 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

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.

Figure 2. Back-transformed mean (±SEM) consump-tion (g per patty) of four identical palatable 10 % pollen treatment diets. Trial duration was 4 days, and 16 rep-licate 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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3.4. Experiment 4—the phagostimulatory effects of bee bread and fresh pollen

There was a significant effect of diet on con-sumption (F2,89=116.36, P <0.001) (Figure 5).

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 predom-inantly 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).

3.5. Experiment 5—phagostimulant effect on free-flying colonies

There was again a significant effect of diet on consumption (F2,40=28.33, P <0.001) (Figure6).

There was a significant difference between the

Figure 4. Cumulative mean mass loss (±SEM) per con-trol patty over the 10-day period within the incubator (n =32).

Figure 5. Back-transformed mean (±SEM) consump-tion (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).

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consumption of all three test diets: control and pollen extract diet (P <0.001), control and extract-ed 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 con-sumption (F3,56=4.71, P =0.01) (Figure7). 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 differ-ence in the mean consumption of the diet contain-ing the hexane extract only and the diet containcontain-ing the ethyl acetate extract only (P =0.85) or the diet containing the methanol extract only (P =0.06).

There was no significant difference in the con-sumption of the diet containing ethyl acetate ex-tract only and the diet containing methanol exex-tract only (P =0.30).

4. DISCUSSION

Experiments 1 and 2 showed that regular dis-tribution of treatments above the brood within each hive adequately reduced any bias from spa-tial variation of the brood below. The method did not produce false positives, and it correctly distin-guished 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 approxi-mate colony temperature could be replicated, there was no facility to replicate colony humidity. Increased colony humidity is almost certainly go-ing 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 consump-tion by bees.

The experiment assessing the phagostimulato-ry effects of bee bread and fresh pollen (experi-ment 4) showed that bees exhibited no significant preference for diets containing either freshly for-aged 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 in-creased 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 ob-served here.

The lack of a preference for either diet may also reveal additional information relating to the chem-ical nature of phagostimulants in pollens. Considering the function of phagostimulants may be important when devising identification

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methods. Volatiles are easily analysed using gas chromatography, but less volatile, more polar, compounds are likely to require the use of alter-native analytical methods in their identification. If phagostimulants are volatile, functioning via ol-factory 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 (Schmidt1985).

The experiment assessing the strength of ex-tractable 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 sum-mer. The consumption of the pollen extract diet was approximately five times greater than the control, while the extracted pollen diet was con-sumed 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 consump-tion 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 ob-served mean consumption values themselves

may suggest a trend in the phagostimulatory ac-tivity of extracts of n -hexane extract < ethyl ace-tate extract < methanol extract < combined ex-tracts. No other examples of multiple-choice feed-ing trials directly comparfeed-ing the activity of differ-ent polarity solvdiffer-ent extracts have been found, but these data appear to indicate that the more active phagostimulants reside in more polar solvent ex-tracts. 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 Hanna2006), potential-ly 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 chal-lenges. 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 sup-plemental diets within these most recent studies vary but are comparable to some of those previ-ously observed. The colonies used within these studies were free-flying field colonies, with no restrictions placed upon them. Although it is per-haps preferable to conduct such experiments using large numbers of colonies, this within-hive repli-cation method offers a viable alternative, should financial or infrastructural constraints prevent the use of more colonies.

ACKNOWLEDGMENTS

This research was supported by the Perry Founda-tion and the British Beekeepers AssociaFounda-tion (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.

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Apis mellifera / régime alimentaire / nourrissage / approvisionnement

Ein Biotest zur Untersuchung von phagostimulatorischen Effekten von Pollen, Bienenbrot und Pollenextrakten in frei fliegenden Bienenvölkern.

Apis mellifera / Phagostimulantien / Diäten / Fütterung / Sammeln

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