Honey bee colonies change their foraging decisions after in-hive experiences with unsuitable pollen.

Pollen is the protein resource for Apis mellifera and its selection affects colony development and productivity. Honey bee foragers mainly lose their capacity to digest pollen, so we expect that those pollen constituents that can only be evaluated after ingestion will not influence their initial foraging preferences at food sources. We predicted that pollen composition may be evaluated in a delayed manner within the nest, for example, through the effects that the pollen causes on the colony according to its suitability after being used by in-hive bees. 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 (Diplotaxis tenuifolia). Dual-choice experiments revealed that foragers did not avoid adulterated pollens at the foraging site; however, they avoided pollen that had been offered adulterated within the nest on 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.

Differences in olfactory sensitivity and odor detection correlate with foraging task specialization in honeybees Apis mellifera.

Division of labor is central to the ecological success of social insects. Among honeybees foragers, specialization for collecting nectar or pollen correlates with their sensitivity to gustatory stimuli (e.g. sugars). We hypothesize that pollen and nectar foragers also differ in their sensitivity to odors, and therefore in their likelihood to show odor-mediated responses. To assess foragers sensitivity to natural odors, we quantified the conditioning of the proboscis extension reflex (PER) to increasing concentrations (0.001; 0.01; 0.1; 1 M) of linalool or nonanal. Furthermore, we compared electroantennogram (EAG) recordings to correlate bees' conditioned responses with the electrophysiological responses of their antennae. To further explore differences of the antennal response of foragers in relation to task-related odors, we registered EAG signals for two behaviorally ''meaningful'' odors that mediate pollen collection: fresh pollen odors and the brood pheromone (E)-ß-ocimene. Pollen foragers performed better than nectar foragers in PER conditioning trials when linalool and nonanal were presented at low concentrations (0.001, 0.01 M). Consistently, their antennae showed stronger EAG signals (higher amplitudes) to these odors, suggesting that differences in sensitivity can be explained at the periphery of the olfactory system. Pollen and nectar foragers detect pollen odors differently, but not (E)-ß-ocimene. Pollen volatiles evoked EAG signals with hyper and depolarization components. In pollen foragers, the contribution of the hyperpolarization component was higher than in nectar foragers. We discuss our findings in terms of adaptive advantages to learn subtle olfactory cues that influence the ability to better identify/discriminate food sources.

Young honeybees show learned preferences after experiencing adulterated pollen.

Pollen selection affects honeybee colony development and productivity. Considering that pollen is consumed by young in-hive bees, and not by foragers, we hypothesized that young bees learn pollen cues and adjust their preferences to the most suitable pollens. To assess whether young bees show preferences based on learning for highly or poorly suitable pollens, we measured consumption preferences for two pure monofloral pollens after the bees had experienced one of them adulterated with a deterrent (amygdalin or quinine) or a phagostimulant (linoleic acid). Preferences were obtained from nurse-aged bees confined in cages and from nurse bees in open colonies. Furthermore, we tested the bees' orientation in a Y-maze using a neutral odour (Linalool or Nonanal) that had been previously associated with an amygdalin-adulterated pollen. Consumption preferences of bees, both in cages and in colonies, were reduced for pollens that had been adulterated with deterrents and increased for pollens that had been supplemented with linoleic acid. In the Y-maze, individuals consistently avoided the odours that they had previously experienced paired with the deterrent-adulterated pollen. Results show that nurse-aged bees associate pollen-based or pollen-related cues with either a distasteful/malaise experience or a tasty/nutritious event, leading to memories that bias their pollen-mediated response.

Selective recruitment for pollen and nectar sources in honeybees.

Honeybees (Apis mellifera) use cues and signals to recruit nestmates to profitable food sources. Here, we investigated whether the type of resource advertised within the colony (i.e. pollen or nectar) correlates with the choices of recruits at the feeding site. We observed that pollen recruits preferred to collect pollen once arrived for the first time at the feeding site, while nectar recruits preferred to forage sucrose solutions. Bees recruited by foragers carrying both resources showed intermediate preferences. Studying the plasticity of this response, we found that nectar recruits have a low probability of switching to pollen collection, yet pollen recruits were likely to switch to sucrose solution of increasing concentrations. Our results show that cues associated with the advertised resource type correlate with the foraging tendency of recruits for pollen and sucrose solution, a feature that would guarantee an efficient resource collection.

Correlation between octopaminergic signalling and foraging task specialisation in honeybees.

Regulation of pollen and nectar foraging in honeybees is linked to differences in the sensitivity to the reward. Octopamine (OA) participates in the processing of reward-related information in the bee brain, being a candidate to mediate and modulate the division of labour among pollen and nectar foragers. Here we tested the hypothesis that OA affects the resource preferences of foragers. We first investigated whether oral administration of OA is involved in the transition from nectar to pollen foraging. We quantified the percentage of OA-treated bees that switched from a sucrose solution to a pollen feeder when the sugar concentration was decreased experimentally. We also evaluated if feeding the colonies sucrose solution containing OA increases the rate of bees collecting pollen. Finally, we quantified OA and tyramine (TYR) receptor genes expression of pollen and nectar foragers in different parts of the brain, as a putative mechanism that affects the decision-making process regarding the resource type collected. Adding OA in the food modified the probability that foragers switch from nectar to pollen collection. The proportion of pollen foragers also increased after feeding colonies with OA-containing food. Furthermore, the expression level of the AmoctαR1 was upregulated in foragers arriving at pollen sources compared with those arriving at sugar-water feeders. Using age-matched pollen and nectar foragers that returned to the hive, we detected an upregulated expression of a TYR receptor gene in the suboesophageal ganglia. These findings support our prediction that OA signalling affects the decision in honeybee foragers to collect pollen or nectar.

Pollen reinforces learning in honey bee pollen foragers but not in nectar foragers.

Searching for reward motivates and drives behaviour. In honey bees Apis mellifera, specialized pollen foragers are attracted to and learn odours with pollen. However, the role of pollen as a reward remains poorly understood. Unlike nectar, pollen is not ingested during collection. We hypothesized that pollen (but not nectar) foragers could learn pollen by sole antennal or tarsal stimulation. Then, we tested how pairing of pollen (either hand- or bee-collected) and a neutral odour during a pre-conditioning affects performance of both pollen and nectar foragers during the classical conditioning of the proboscis extension response. Secondly, we tested whether nectar and pollen foragers perceive the simultaneous presentation of pollen (on the tarsi) and sugar (on the antennae) as a better reinforcement than sucrose alone. Finally, we searched for differences in learning of the pollen and nectar foragers when they were prevented from ingesting the reward during the conditioning. Differences in pollen-reinforced learning correlate with division of labour between pollen and nectar foragers. Results show that pollen foragers performed better than nectar foragers during the conditioning phase after being pre-conditioned with pollen. Pollen foragers also performed better than nectar foragers in both the acquisition and extinction phases of the conditioning, when reinforced with the dual reward. Consistently, pollen foragers showed improved abilities to learn cues reinforced without sugar ingestion. We discussed that differences in how pollen and nectar foragers respond to a cue associated with pollen greatly contribute to the physiological mechanism that underlies foraging specialization in the honeybee.