Honeybee colonies change their foraging decisions after in-hive experiences with unsuitable pollens

doi:10.21203/rs.3.rs-2578515/v1

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Honeybee colonies change their foraging decisions after in-hive experiences with unsuitable pollens

https://doi.org/10.21203/rs.3.rs-2578515/v1

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Pollen is the protein resource for honeybees (Apis mellifera) and its selection affects colony development and productivity. Foragers do not consume pollen at the food source, so we expect pollen components evaluated post-ingestively not to bias their initial foraging preferences. Alternatively, pollen composition could be assessed in a delayed manner, by experiences with the pollens being processed inside the nest or with their effects on the colony. To address whether pollen foraging is mediated by in-hive experiences, we conducted dual-choice experiments to test the avoidance of pollen adulterated with amygdalin, a deterrent that causes post-ingestion malaise. In addition, we recorded pollen selection in colonies foraging in the field after being supplied or not with amygdalin-adulterated pollen from one of the dominant flowering plants (D. tenuifolia). Dual-choice experiments revealed that foragers did not avoid adulterated pollens at the foraging site; however, they could avoid unadulterated pollens that had been offered adulterated within the nest the previous days. In field experiments, pollen samples from colonies supplied with amygdalin-adulterated pollen were more diverse than controls, suggesting that pollen foraging was biased towards novel sources. Our findings support the hypothesis that pollen assessment relies on in-hive experiences mediated by pollen that causes post-ingestive malaise.

Biological sciences/Zoology

Biological sciences/Zoology/Animal behaviour

Biological sciences/Zoology/Animal physiology

Biological sciences/Ecology/Behavioural ecology

adulterated pollen

amygdalin

forager bees

foraging preferences

in-hive experiences

pollen assessment

Animals depend on their perceptual ability to select the most suitable food resources to obtain nutrients. To this end, foragers assess the resources in situ to decide whether to exploit them or not. Furthermore, foraging occurs not only to satisfy the immediate needs of foragers, but of other group members (1). Then, resource selection can be conflicting if the requirements of the individuals foraging and using the resources is not the same. Among social insects, foraging is restricted to a subset of workers whose requirements differ from other colony mates (2). For example, in the honeybee Apis mellifera L., pollen is not directly used by foragers, but by nurse bees that consume most of the pollen and process it to feed the larvae and the rest of the colony mates (3, 4). So far, little is known to what extent pollen selection is based on foragers’ assessment at the foraging site, or it is influenced by pollen-related experiences within the nest.

Pollen is the main protein and lipid resource for honeybee colonies and could strongly differ in composition among plant species (5, 6, 7). Pollen might also contain deterrent compounds, such as nicotine alkaloid in the tobacco plant or the cyanogenic glycoside amygdalin in almond pollen (8, 9). Honeybee foragers (workers over 2 weeks of age) collect pollen with their legs and mouthparts at the food source but do not ingest it, as they are no longer capable of digesting pollen (3). In this scenario, foragers are unlikely to evaluate pollen composition at the source since detection of many of its constituents requires ingestion. Evidence suggests that foragers' assessment of pollen is limited to pre-ingestive pollen cues available in the outermost layer of the grain (10), the pollenkitt. The pollenkit also enables foragers to discriminate between resources from different flowering plant species (11, 12) and it might provide information about pollen constituents as its composition could be similar to that of the internal fractions of pollen (13). Consistently, research on learning and memory shows that pollen reinforcement occurs without pollen ingestion, but through chemo-tactile stimulation of the bee's tarsi or antennae (14).

Inside the nest, young workers process pollen into bee bread. Pollen processing includes the addition of sugars, honey and glandular secretions that make the resource easier to digest and probably increase its attractiveness to the bees (15). Nurse bees (in-hive workers of about 3–10 days of age) consume both fresh pollen and bee bread to incorporate protein into their hypopharyngeal gland secretions that are used for feeding larvae, young workers and queens (4). Nurses also share jelly with foragers (16) providing them with protein and likely, with information on the nutritional quality of the colony's diet. However, it is unknown whether species-specific information of pollens can also be shared through this mechanism.

During the food selection process, avoiding intoxication is as important as achieving nutrition. Interestingly, nursed-aged bees avoid consuming pollens that have been experienced as unsuitable (e.g., adulterated with amygdalin or quinine), which suggests that they participate in the process of pollen selection inside the nest (17). So far, whether and to what extent foragers are able to avoid unsuitable resources solely based on their ability to assess pollen composition at the sources or as a consequence of information acquired within the nest (18,19), remains to be tested. We hypothesise that foraging preferences are adjusted after experiences mediated by the pollens that are being processed inside the nest. As nurses do, foragers might learn to reject pollen cues directly after ingesting small samples of unsuitable pollen stored in cells (20, 21, 22). Alternatively, they could associate pollen-based cues with the modifications that in-hive bees might generate on the nest environment (23) for example, because of the accumulation of unsuitable pollens in hive stocks if it is less consumed by nurse bees.

To unveil how foragers obtain information to guide pollen searching, we investigated their abilities to perceive, learn and memorise pollen cues according to the resource quality and in-hive experiences. In dual-choice experiments with colonies confined in flying cages, we first evaluated foragers´ avoidance response to pollens adulterated with amygdalin at the food source. In a second experiment, we quantified preferences for two unadulterated pollens, after one of them was offered adulterated inside the nest. In a third experiment we measured pollen selection in the field by means of the diversity of pollen samples trapped at the entrance of the hives that were supplied (or not) with amygdalin-adulterated pollen from D. tenuifolia, one of the dominant flowering plants in the surroundings. How pollen foragers obtain information for pollen selection is relevant and timely given the increasing alteration of environments by humans that challenges the ability of bees to obtain an adequate diet.

Study site and bees

We performed experiments during the summer seasons of 2019/2020 and 2020/2021. Experiments I and II were done in the Experimental Field of the School of Exact and Natural Sciences of the University of Buenos Aires (34°32′S, 58°26′W). We used 8 colonies for experiment I, 8 for experiment IIA and 8 for experiment IIB. All colonies contained a mated queen, 3–4 brood frames, and 1–2 frames with food reserves. For these experiments, we confined colonies in flying cages (9m x 3m x 3m) one at a time. The enclosure prevented the interference of foragers with bees from other colonies, while it limited the sources of pollen and artificial nectar available to the experimental colony. Once in the flying cage, we trained foragers to visit a foraging station offering crushed bee-collected multifloral pollen in an ad libitum feeder. Foraging station was located 8m from the hive entrance. The colonies were confined in the cages for approximately one week prior to the onset of the experiments, which was the minimum time necessary to get a constant and relatively high number of pollen foragers visiting the station.

Experiment III was performed in a sunflower crop, located in Coronel Pringles, Buenos Aires, Argentina (68°6'11.94"S – 61°25'46.00"O). We used 22 colonies randomly chosen from a total of 68 colonies settled in 3 different plots within the field. All colonies contained a mated queen, 7–8 brood frames, and 2 frames with food reserves.

We performed all experiments according to the animal care guidelines of the National Institute of Health (1985) and the current laws of Argentina.

Monofloral pollens and pollen quality modification

For bees to differentiate pollen according to their related cues (odour, tastes, texture, etc.) and for preferences among pollen options to be comparable within and between experiments, we used monofloral pollens. Monofloral pollens were obtained from multifloral or monofloral bee-collected samples (provided and characterised by Pampero, Amuyen, and CoopSol cooperatives). In the case of multifloral samples, we separated the pollen pellets according to their botanical origin based on their colour, brightness, texture and degree of agglutination. Most pollen samples were obtained in the same season used for data collection. To reduce the quality of pollen, we crushed, weighted and mixed the monofloral pollen with amygdalin (0.1M) (17) (Sigma-Aldrich, BioXtra, ≥ 97.0%), a cyanogenic glycoside that naturally occurs in almond pollen (8, 9). Amygdalin causes post-ingestional mortality when added to a sugar solution fed to the bees (24).

Experiment I: Testing foragers´ preferences for unadulterated vs. amygdalin-adulterated pollen at the food source

The aim of experiment I was to test whether foragers were able to assess pollen composition at the foraging site, hence, to avoid the amygdalin-adulterated pollen during their foraging visits. To this end, we measured foraging preferences of colonies for two different monofloral-pollen: one offered adulterated and the other unadulterated. Such preferences were compared with preferences measured in a different group of colonies, whose foragers chose between the same two monofloral pollens when both were offered unadulterated (Fig. 1A).

Here, we used Schinopsis vs. Salix pollens. At the foraging station, Schinopsis pollen was always offered unadulterated (Fig. 1A). On the other hand, Salix could be presented adulterated or unadulterated according to the treatment assigned to the colony. The two pollens were offered in separate feeders located next to each other (2cm apart). The pollen feeders consisted of 360ml opaque plastic pots with 8 holes (7mm ⦽) in the top (lid) through which the bees could enter and leave with pollen loads. Inside the pots, 2-3g of pollen were present attached to the bristles of a pipe cleaner (20cm) rolled into a spiral shape (25). The design of these feeders favours the use of pollen olfactory cues by foragers while it limits the range of visual cues.

The bees that left the feeders loaded were counted and captured to ensure that they do not carry pollen back to the nest. Pollen foragers had no previous experience with the pollen offered during testing, so we measured preferences that were not influenced by the experience.

The experiment I consisted of 2 testing events on successive days (24hr interval): the first one (T0) with both pollens unadulterated and the second one (T1) with Salix offered either adulterated or not (Fig. 1A). Although all experimental hives came from the same stock and the same apiary, we do not rule out that there may be variability between colonies, probably related to the different types of pollens they experienced before confinement or the particular nutritional needs of the colonies. Testing trials were carried out at 11:00am. Trials were extended until we measured the preferences of at least 30 bees or until 1 hour had elapsed. From each test (T0 and T1) we obtained the proportion of foragers that preferred Salix as the number of bees foraging on Salix over the number of total foragers collecting pollen in both feeders. We used preferences obtained at T0 as covariate in the analysis (see Statistic Analysis). Then, we compared colony preferences for unadulterated- or adulterated Salix pollen presented at the foraging site.

Experiment II: Testing foragers´ preferences after the offering of amygdalin-adulterated pollen inside the hive

Here we addressed whether preferences of colonies that had experienced adulterated pollen differed from preferences of colonies that had experienced the same pollen unadulterated.

The experiments consisted of 2 phases and 3 testing events (Fig. 1B and 1C). In a first test (T0) we measured initial preferences for two monofloral pollens. Again, initial preferences were used as covariate in the analysis to account for variations among experimental colonies (see Statistic Analysis). Immediately after T0, we ran the first phase of the experiment (P1) lasting two days, when we offered the adulterated or unadulterated pollen that would establish the in-hive experience. In experiment IIA, we offered adulterated or unadulterated Salix pollen at the foraging station and let the bees collect and introduce it into the nest. Here, Salix pollen was tested against Schinopsis sp pollen, which was always presented unadulterated (Fig. 1B). In experiment IIB, adulterated or unadulterated Brassica napus pollen was hydrated to a bee bread-like paste that we poured directly inside the hive (10ml each day for two days), on the top of its central frames. At the foraging station B. napus pollen was tested against and Diplotaxis tenuifolia pollen, both unadulterated (Fig. 1C). Note that, despite being similar, experiment IIA includes the possibility that bees foraging on adulterated pollen in P1 (Fig. 1) may establish aversive associations with the resource over successive visits.

Two days after the onset of the experiment, we measured the effect of in-hive experience on preferences for pollens (both unadulterated) on a second testing trial (T1; Fig. 1B). We then conducted the second phase of the experiment (P2), which ran from day 2 to day 4. In the second phase of the experiment IIA, we let the foragers naturally take the unadulterated Salix pollen from the station back to the nest to assess whether memories established with unsuitable pollens can be reverted on the basis of newly available information. In the second phase of experiment IIB, we also let the foragers take the unadulterated B. napus pollen back to the nest, but at the same time we offered it adulterated inside the hive for the second time. By doing this, we tested the extent to which in-hive pollen mediated experiences resist conflicting information. On day 4, we tested foraging preferences for the third time (T2; Fig. 1C). Proportion of bees at testing trials were compared between colonies treated with unadulterated- and adulterated-pollen.

Experiment III: Testing pollen selection after the offering of amygdalin-adulterated pollen inside the hive

Here, we investigated whether in-hive experiences mediated by unsuitable pollen affected pollen selection of commercial hives that foraged in an agricultural setting. To this end we supplied a first group of colonies with amygdalin-adulterated pollen of D. tenuifolia, a dominant flowering plant highly available in the surrounding prior and during the first day of the experiment. In addition, a second group of colonies was supplied with unadulterated D. tenuifolia pollen. Finally, a third group of colonies received no extra pollen. We offered D. tenuifolia pollen like in exp. IIB.

We used the floral diversity of collected pollen as a proxy of the colony's pollen selection. Pollen samples were collected using conventional pollen traps (frontal-entrance trap), consisting of a wooden structure with a removable metal mesh inside. We sampled colonies for 4 hours, one day before (T0) and two days after the beginning of in-hive pollen-offering (T1; Fig. 1D). We separated pollen pellets from the samples based on their colour. We weighted them to calculate the relative proportion of each pollen type and in turn, we used those proportions to calculate the Shannon Diversity Index (26). We used the diversity of samples taken at T0, as a covariate to control for differences among colonies before manipulation.

Statistical analysis

All statistical analyses were performed in R (27). Differences in forager choices (proportions) of experiment I were addressed by means of generalised linear model (GLM) and in experiment II, differences were addressed by means of generalised linear mixed model (GLMM), both experiments with binomial distributions (28). Models´ assumptions were checked with the DHARMA library (29) and the variation in the number of individuals tested in each event was accounted for through the weights. In experiment I, we explored the impact of treatment (unadulterated and adulterated) as a fixed effect and we added initial preferences as covariate. Here, we used the glm function of the Stats package. In experiments II (IIA and IIB) we analysed the effect of treatment (unadulterated and adulterated) and test (T1 and T2) as fixed effects and its interaction. We added the colony and the month when the colony was studied, as random factors and the initial preferences as covariate. Here, we used the glmer function of the lme4 package (30) that uses Wald Z-tests to approximate p-values for GLMMs (31). Post hoc contrasts were conducted on models to assess effects and significance between fixed factors using the “emmeans” function of the ‘emmeans’ package version 1.4 (32) with a significance level of 0.05.

Differences in the Shannon Diversity Index (H) of experiment III were assessed using general linear models (GLM) with normal distribution. We explored the effect of treatment (unadulterated, adulterated, no-pollen) and we used initial preferences as covariate. Homoscedasticity and normality assumptions were checked (Levene and Shapiro-Wilk tests, respectively). Tukey’s tests were used for contrasts using the multicomp package in R (33).

Experiment I: Testing foragers´ preferences for unadulterated vs. amygdalin-adulterated pollen at the food source

Colonies did not change their foraging preferences when amygdalin was added to the pollen at the feeder. Then, our results showed that foraging preferences of colonies exposed to adulterated (0.567 ± 0.025) or unadulterated pollen (0.586 ± 0.026) were similar (Treatments: Chiq = 0.291, df: 1, P = 0.590; Fig. 2).

Experiment II: Testing foragers´ preferences after the offering of amygdalin-adulterated pollen inside the hive

Colonies significantly reduced their foraging preferences for the pollen that had been offered adulterated inside the nest, either if it was incorporated by foragers themselves (exp. IIA; Treatment*Time Chisq = 12.606, df = 1, P < 0.001; Fig. 3A) or artificially, on the top of the frames (exp. IIB, Treatment: Chisq = 17.999, df = 1, P < 0.001; Treatment*Test: Chisq = 0.076, df = 1, P = 0.782; Fig. 3B). Both experiments (IIA and IIB) showed similar results for T1. In experiment IIA, foragers from amygdalin-treated colonies showed a lower preference for Salix pollen (adulterated: 0.479 ± 0.049, N = 4) than bees from unadulterated-pollen treated colonies (0.682 ± 0.043, N = 4) (unadulterated - adulterated: z-ratio = 3.023; P = 0.003). Similarly, in experiment IIB, bees from amygdalin-treated colonies choose B. napus pollen less (adulterated: 0.255 ± 0.049, N = 4) than controls (unadulterated: 0.459 ± 0.055, N = 4) (unadulterated - adulterated: z-ratio = 3.387, P < 0.001).

The results of the experiments IIA and IIB did differ by the third testing event (T2). In experiment IIA, where foragers switched from carrying adulterated to carrying unadulterated Salix pollen, differences detected at T1 between amygdalin-treated colonies and controls were lost (adulterated: 0.565 ± 0.049; unadulterated: 0.523 ± 0.050; unadulterated - adulterated: z-ratio =-0.608, P = 0.543). In contrast, when we let the foragers incorporate unadulterated B. napus pollen while we still offered it pollen inside the hive (experiment IIB), preference for B. napus pollen remained lower in the treated than in the control group (unadulterated: 0.495 ± 0.062; adulterated: 0.265 ± 0.049; unadulterated - adulterated: z-ratio = 3.405, P < 0.001).

Experiment III: Testing pollen selection after the offering of amygdalin-adulterated pollen inside the hive

Colonies showed differences in the diversity (i.e., composition and/or relative abundance) of pollens selected after the addition of adulterated pollen inside the hives (Treatments: F_2;17 = 4.671; P = 0.024, Fig. 4). Colonies that did not receive pollen showed the lowest diversity index (no-pollen: 1.1 ± 0.29, N = 6) while colonies that received adulterated pollen showed the highest index value (1.88 ± 0.5, N = 8). Intermediate values of diversity were observed in colonies that received unadulterated pollen (1.43 ± 0.59, N = 7). Statistical analysis detected significant differences in the H-index between colonies treated with adulterated and colonies with no added pollen (adulterated - no pollen: t-ratio = -2.584; P = 0.048), but not between the colonies with no added pollen and colonies supplied with unadulterated pollen (no-pollen - unadulterated: t-ratio = -0.976; P = 0.601) or between colonies supplied with unadulterated and amygdalin-adulterated pollen (unadulterated - adulterated: t-ratio = -1.725; P = 0.225).

Many questions about the process by which honeybees evaluate pollen and how such evaluation drives foraging decisions remain unanswered (34). Here we ask how bee colonies deal with deterrents in pollen. We found that foragers do not respond to the addition of amygdalin at the pollen source during the first visits. Instead, the offering of amygdalin-adulterated pollen within the hive was enough to change bees´ foraging preferences.

Given the life history of a highly social insect, the loss of the reproductive phase of Apis mellifera workers caused a drastic reduction in pollen intake (essential for ovary development) in the mature stages (35), which could have affected their ability to fully evaluate the resource. Thus, to avoid toxins that can only be detected after ingestion of resources, honeybee colonies might rely on experiences with the resource inside the hive to adjust the choice of pollens outside. The impact of such experiences on foraging have been well documented among social insects. It has been shown in honeybees (e.g. 36), stingless bees (37), bumblebees (38), yellow jacket (39); and ants colonies (40) within both appetitive (41, 42) and aversive contexts (43, 40).

Pollen is a complex and chemically variable resource that provides the nutrients for the development of the colony. Pollens can differ greatly in composition depending on their botanical origin, with some pollen types containing low levels of protein and/or lacking essential nutrients (44, 45, 46, 47, 48, 7). Pollen collection is therefore expected to be biased towards pollen with higher major nutrient content, such as protein or lipids. Preferences for protein-rich pollen have been observed in other bee species (49), but not in the honeybee (50, 51, 52, 53, 54, 55, 56, 10). In line with honeybees' limitations to perceive certain compounds in pollen, bees did not respond to the amygdalin at the foraging site. Results suggest that bees did not avoid amygdalin itself, but pollen-related cues associated with the post-ingestive malaise that amygdalin causes. In an additional experiment, we excluded the possibility that pollen hydration could facilitate amygdalin detection at the food source, as forager also failed to spontaneously avoid adulterated pollen that was offered as paste (Fig S1).

So far, our findings support the hypothesis that honeybees respond to amygdalin after ingestion. The idea that amygdalin causes malaise experiences post ingestion was proposed by observing that bees olfactory conditioned with a sucrose-amygdalin solution performed as good as controls during the first trials but stopped responding to the odour in the followings (24, 57, but see 58). Delayed aversion caused by amygdalin can explain why foragers failed to avoid collecting the unsuitable resource on their first visit to the source and why they succeeded in changing their preferences after the offering of adulterated pollen inside the nest.

Changes in pollen foraging preferences after in-hive experiences might involve learning of pollen-based cues (odour, taste, texture, etc.) with the post-ingestive consequences of amygdalin consumption (57, see also 24). In line with a process mediated by associative memories, we observed that avoidance response triggered by adulterated pollen was reversible upon the incorporation of the same resource but suitable. So far, how foragers learn about the unsuitable pollen remains to be investigated. Our results do not rule out that foragers could have learnt the association along the successive visits to pollen sources containing amygdalin (exp. IIA). However, repeated experiences with adulterated pollen at the food source appear not to enhance avoidance of the unsuitable pollen compared to experiences taking place entirely within the nest (exp. IIB).

Other than in the food source, foragers might learn about pollen unsuitability directly from adulterated pollens inside the hive while they inspect cells assessing nest stocks, for example if they consume small amounts of pollen that is being processed into bee bread (20, 21, 22). Newly prepared bee bread is easily digestible and more attractive to the bees than raw pollen and stored pollen reserves (15). Furthermore, the bee bread is likely to maintain pollen cues, allowing foragers to associate them with the consequences of its consumption. Early evaluation of pollen while being processed could be adaptive, informing the bees about the suitability of the pollens available in the surroundings and preventing foragers from collecting and accumulating resources that are harmful to larvae and young bees.

Alternatively, foragers might learn pollen through modifications that other mates generate in the nest environment (23). In this regard, we recently showed that nurse bees are sensitive to both toxic and nutritional pollen components. Nurse-aged bees could associate pollen-based or pollen-related cues with malaise experiences triggered by pollens adulterated with amygdalin or quinine, leading to aversive memories that reduce pollen consumption and discourage orientation to pollen-related cues (17). If this is the case, choices of the young bees may constitute a second step in the pollen selection process, whereby unsuitable pollen would be less consumed and probably accumulated in the nest providing information for the rest of the colony mates. For example, volatiles or tastes of unsuitable pollen that accumulate, age, and/or become "entombed" in the cells (59) may induce latent inhibition among foragers that translates such experiences into avoidance responses during searching (60). In addition, we must consider the impact of direct interactions between foragers and in-hive bees who, having learned about the noxious properties of certain pollens, may discourage foragers e.g., not following their dances, or not sharing the food they begged.

The hypothesis that pollen decision making is influenced by the access to the information about the different resources being processed in the nest is supported by an additional experiment with colonies deprived of pollen intake for 3 consecutive days (Fig S2). Using hives with permanent pollen traps that were fed artificial protein supplements, we observed a decrease in the pollen diversity of their samples compared with colonies that freely incorporated pollen (Fig S2). We speculate that the lack of information about the different pollens inside the nest, led foragers to make decisions based on their innate preferences for pollen-related cues rather than on the effects of pollen on the nutritional/health status of the colony.

On the contrary, foraging patterns of the colonies supplied with amygdalin-adulterated pollen (exp. III) showed a shift towards diversification of pollen sources. This finding is consistent with evidence that colonies exposed to pollens low in protein content, lacking essential nutrients and/or containing toxins increase the diversity of pollen types/species they collected (61, 62), likely as a strategy to dilute the effect of the unsuitable resource by mixing different pollens (44, 45, 46, 7). In addition, colony responses to nutritional deficits include the selection of pollens with complementary nutrients (18, 19).

Diversification might be achieved by reallocation of foragers to novel resources, likely induced by the avoidance response to pollens that had been experienced as unsuitable inside the hive. We detected no changes in the total weight of pollen samples collected in colonies treated with no-pollen, unadulterated or adulterated pollen (Fig S3) suggesting that pollen foraging activity did not change after the offering of unsuitable pollen. This evidence rules out that diversification is due to a generalised increase in foraging activity that enables the incorporation of pollen from less represented floral species in the colony's diet because of a better exploration/exploitation of the environment.

In an attempt to measure changes in the proportion of D. tenuifolia pollen collected after being offered adulterated within the colonies, we noted important fluctuations in the availability of D. tenuifolia pollen during the experimental period that prevented detecting any effect among treatments. D. tenuifolia, was abundant during the first day of the experiment (28 January 2021), however, its availability decreased during the following days (31 January onwards), resulting in very little D. tenuifolia pollen in the pollen samples of all treatments (Fig S4A). Interestingly, a preliminary study conducted in 2020 (January 11 to 15), where D. tenuifolia remained abundant during all the experimental period revealed that amygdalin-treated colonies showed a reduction in the proportions of D. tenuifolia collected compared to control colonies (Fig S4B).

In the 2020 season, colonies treated with amygdalin-adulterated pollen that exhibited the reduction in the proportion of D. tenuifolia, also exhibit a decrease in the diversity of their pollen samples (Fig S5), suggesting that despite differences in experimental conditions between confined hives (collecting only two pollen inside the flying cage; exp 2) and hives collecting freely in the field (exp 3), colony´s response to the incorporation of unsuitable pollens may be the same. Furthermore, this approach was complemented using a third group of colonies treated with pollen adulterated by quinine, a naturally bitter-tasting alkaloid that bees detect (and reject) prior to ingestion [57, 17]. Thus, we confirmed that the changes in pollen selection patterns not only occur with amygdalin-adulterated pollen but extends to pollens adulterated with other aversive compounds that may act before or after ingestion.

Altogether our results highlight the importance of resource-mediated experiences within the social environment of the hive and goes further suggesting that social learning, i.e., the learning that occurs within the social context of the colony [6–8]) is critical for selecting resources whose constituents cannot be perceived at the food source. With increasing land use and extensive production practices that threaten the diversity of resources used by the bee, our finding is timely and opens the door for further research into the mechanisms and strategies that honeybees have for selecting their pollen sources.

Acknowledgements

We thank M. J. Corriale for her help with statistical analyses and W. Farina for the fruitful comments at the early stage of this paper. Thanks to L. Martinez for his collaboration in the field experiments. We also thank the anonymous reviewers for their positive comments and suggestions.

Funding.

This study was partly supported by grants from Agencia Nacional de Promoción Cientıfica y Tecnológica (PICT_2017-2688) to A. Arenas

Data availability statement.

All data generated or analysed during this study are included in this published article [and its supplementary information files].

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Honeybee colonies change their foraging decisions after in-hive experiences with unsuitable pollens

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Abstract

Figures

Introduction

Materials And Methods

Study site and bees

Monofloral pollens and pollen quality modification

Experiment I: Testing foragers´ preferences for unadulterated vs. amygdalin-adulterated pollen at the food source

Experiment II: Testing foragers´ preferences after the offering of amygdalin-adulterated pollen inside the hive

Experiment III: Testing pollen selection after the offering of amygdalin-adulterated pollen inside the hive

Statistical analysis

Results

Experiment I: Testing foragers´ preferences for unadulterated vs. amygdalin-adulterated pollen at the food source

Experiment II: Testing foragers´ preferences after the offering of amygdalin-adulterated pollen inside the hive

Experiment III: Testing pollen selection after the offering of amygdalin-adulterated pollen inside the hive

Discussion

Declarations

Acknowledgements

References

Additional Declarations

Supplementary Files

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