Honey bees can store and retrieve independent memory traces after complex experiences that combine appetitive and aversive associations

Real-world experiences do often mix appetitive and aversive events. Understanding the ability of animals to extract, store and use this information is an important issue in neurobiology. We used honey bees as model to study learning and memory after a differential conditioning that combines appetitive and aversive training trials. First of all, we describe an aversive conditioning paradigm that constitutes a clear opposite of the well known appetitive olfactory conditioning of the proboscis extension response. A neutral odour is presented paired with the bitter substance quinine. Aversive memory is evidenced later as an odour-specific impairment in appetitive conditioning. Then we tested the effect of mixing appetitive and aversive conditioning trials distributed along the same training session. Differential conditioning protocols like this were used before to study the ability to discriminate odours, however they were not focused on whether appetitive and aversive memories are formed. We found that after a differential conditioning, honey bees establish independent appetitive and aversive memories that do not interfere with each other during acquisition or storage. Finally, we moved the question forward to retrieval and memory expression to evaluate what happens when appetitive and the aversive learned odours are mixed during test. Interestingly, opposite memories compete in a way that they do not cancel each other out. Honey bees showed the ability to switch from expressing appetitive to aversive memory depending on their satiation level.

Honeybee Cognition as a Tool for Scientific Engagement

Apis mellifera (honeybees) are a well-established model for the study of learning and cognition. A robust conditioning protocol, the olfactory conditioning of the proboscis extension response (PER), provides a powerful but straightforward method to examine the impact of varying stimuli on learning performance. Herein, we provide a protocol that leverages PER for classroom-based community or student engagement. Specifically, we detail how a class of high school students, as part of the Ryukyu Girls Outreach Program, examined the effects of caffeine and dopamine on learning performance in honeybees. Using a modified version of the PER conditioning protocol, they demonstrated that caffeine, but not dopamine, significantly reduced the number of trials required for a successful conditioning response. In addition to providing an engaging and educational scientific activity, it could be employed, with careful oversight, to garner considerable reliable data examining the effects of varying stimuli on honeybee learning.

Suitability of drone olfactory sensitivity as a selection trait for Varroa-resistance in honeybees

The most effective strategy against brood diseases, such as those stemming from infestation by the mite Varroa destructor, is the early detection and removal of sick brood. Recent findings suggest that genes associated with worker bee olfactory perception play a central role in Varroa-sensitive hygiene (VSH). In this study, the odour sensitivity of Apis mellifera drones was examined through proboscis extension response (PER) conditioning. Individuals sensitive/insensitive to the two Varroa-parasitised-brood odours (extract-low and extract-high) were used for breeding. Twenty-one queens from a VSH-selected line (SelQ) and nineteen queens from a nonselected line (ConQ) were single-drone-inseminated with sperm from drones that showed either sensitivity (SenD+) or insensitivity (SenD−) to the two extracts. Individual VSH behaviour in a total of 5072 offspring of these combinations (SelQ × SenD+, SelQ× SenD−, ConQ × SenD+, ConQ × SenD−) was subsequently observed in a specially designed observation unit with infrared light. The results from the video observation were also separately examined, considering the genetic origin (VSH-selected or nonselected line) of the participating queens and drones. While the drone PER conditioning results were not significantly reflected in the VSH results of the respective offspring, the genetic origin of the participating queens/drones was crucial for VSH manifestation.

Comparing the Appetitive Learning Performance of Six European Honeybee Subspecies in a Common Apiary

The Western honeybee (Apis mellifera L.) is one of the most widespread insects with numerous subspecies in its native range. How far adaptation to local habitats has affected the cognitive skills of the different subspecies is an intriguing question that we investigate in this study. Naturally mated queens of the following five subspecies from different parts of Europe were transferred to Southern Germany: A. m. iberiensis from Portugal, A. m. mellifera from Belgium, A. m. macedonica from Greece, A. m. ligustica from Italy, and A. m. ruttneri from Malta. We also included the local subspecies A. m. carnica in our study. New colonies were built up in a common apiary where the respective queens were introduced. Worker offspring from the different subspecies were compared in classical olfactory learning performance using the proboscis extension response. Prior to conditioning, we measured individual sucrose responsiveness to investigate whether possible differences in learning performances were due to differential responsiveness to the sugar water reward. Most subspecies did not differ in their appetitive learning performance. However, foragers of the Iberian honeybee, A. m. iberiensis, performed significantly more poorly, despite having a similar sucrose responsiveness. We discuss possible causes for the poor performance of the Iberian honeybees, which may have been shaped by adaptation to the local habitat.

Comparing the appetitive learning performance of six European honeybee subspecies in a common apiary

The Western honeybee (Apis mellifera L.) is one of the most widespread insects with numerous subspecies in its native range. In how far adaptation to local habitats has affected the cognitive skills of the different subspecies is an intriguing question which we investigate in this study. Naturally mated queens of the following five subspecies from different parts of Europe were transferred to Southern Germany: A. m. iberiensis from Portugal, A. m. mellifera from Belgium, A. m. macedonica from Greece, A.m. ligustica from Italy and A. m. ruttneri from Malta. We also included the local subspecies A.m. carnica in our study. New colonies were built up in a common apiary where the respective queens were introduced. Worker offspring from the different subspecies was compared in classical olfactory learning performance using the proboscis extension response. Prior to conditioning we measured individual sucrose responsiveness to investigate whether possible differences in learning performances were due to a differential responsiveness to the sugar water reward. Most subspecies did not differ in their appetitive learning performance. However, foragers of the Iberian honeybee, A. m. iberiensis, performed significantly more poorly, despite having a similar sucrose responsiveness. We discuss possible causes for the low cognitive performance of the Iberian honeybees, which may have been shaped by adaptation to local habitat.

Honey Bees Can Taste Amino and Fatty Acids in Pollen, but Not Sterols

The nutritional composition of food is often complex as resources contain a plethora of different chemical compounds, some of them more, some less meaningful to consumers. Plant pollen, a major food source for bees, is of particular importance as it comprises nearly all macro- and micronutrients required by bees for successful development and reproduction. However, perceiving and evaluating all nutrients may be tedious and impair quick foraging decisions. It is therefore likely that nutrient perception is restricted to specific nutrients or nutrient groups. To better understand the role of taste in pollen quality assessment by bees we investigated nutrient perception in the Western honey bee, Apis mellifera. We tested if the bees were able to perceive concentration differences in amino acids, fatty acids, and sterols, three highly important nutrient groups in pollen, via antennal reception. By means of proboscis extension response (PER) experiments with chemotactile stimulation, we could show that honey bees can distinguish between pollen differing in amino and fatty acid concentration, but not in sterol concentration. Bees were also not able to perceive sterols when presented alone. Our finding suggests that assessment of pollen protein and lipid content is prioritized over sterol content.

The use of A. mellifera drone's olfactory sensitivity towards pathological odours as a selection trait in the breeding against V. destructor

The most effective strategy against brood diseases, such as infestation by the mite Varroa destructor, is the early detection and removal of sick brood. Recent findings suggest that genes associated with worker bees' olfactory perception play a central role in Varroa-sensitive hygiene (VSH). In the following approach, Apis mellifera drones' odour sensitivity was examined through a standardised Proboscis extension response (PER) test. Individuals with a positive/negative conditioning outcome to two parasitised-pupae extracts (extract-low and extract-high) were used for breeding. Twenty-one queens from a VSH selection line (SelQ) and nineteen queens from an unselected line (ConQ) were single-drone-inseminated with drones that showed either a positive (SenD+) or a negative (SenD-) PER test. Individual VSH behaviour of a total of 5072 offspring of these combinations (SelQ x SenD+, SelQ + x SenD-, ConQ x SenD+, ConQ x SenD-) was subsequently observed in a specially designed unit with infrared light. The results from the observation were also separately examined, considering the hygienic status of the participating queens and drones. The results of the PER test of the drones were not significantly reflected in the VSH results of the respective offspring. On the other hand, the participating queens/drones' hygienic status was crucial for the manifestation of VSH.

Prior Experience with Food Reward Influences the Behavioral Responses of the Honeybee Apis mellifera and the Bumblebee Bombus lantschouensis to Tomato Floral Scent

Bee responses to floral scent are usually influenced by both innate biases and prior experience. Honeybees are less attracted than bumblebees to tomato flowers. However, little is known about how tomato floral scent regulates the foraging behaviors of honeybees and bumblebees. In this study, the foraging behaviors of the honeybee Apis mellifera and the bumblebee Bombus lantschouensis on tomato flowers in greenhouses were investigated. Whether the two bee species exhibit different responses to tomato floral scent and how innate biases and prior experience influence bee choice behavior were examined. In the greenhouses, honeybees failed to collect pollen from tomato flowers, and their foraging activities decreased significantly over days. Additionally, neither naïve honeybees nor naïve bumblebees showed a preference for tomato floral scent in a Y-tube olfactometer. However, foraging experience in the tomato greenhouses helped bumblebees develop a strong preference for the scent, whereas honeybees with foraging experience continued to show aversion to tomato floral scent. After learning to associate tomato floral scent with a sugar reward in proboscis extension response (PER) assays, both bee species exhibited a preference for tomato floral scent in Y-tube olfactometers. The findings indicated that prior experience with a food reward strongly influenced bee preference for tomato floral scent.

Exposure to a biopesticide interferes with sucrose responsiveness and learning in honey bees

The entomopathogenic fungus Beauveriabassiana is a widely used biopesticide that is considered as an effective alternative to classical agrochemicals. B. bassiana is thought to be safe for pollinators although little is known about its side-effects on pollinators’ behaviour and cognition. Here, we focused on honey bees and used the proboscis extension response (PER) protocol to assess whether B. bassiana affects individual sucrose responsiveness, non-associative and associative olfactory learning and memory. Fungus-treated bees displayed an enhanced sucrose responsiveness, which could not be explained by metabolic alterations. Strikingly, exposed bees were twice as inconsistent as controls in response to sucrose, showing PER to lower but not to higher sucrose concentrations. Exposed bees habituated less to sucrose and had a better acquisition performance in the conditioning phase than controls. Further, neither mid- nor long-term memory were affected by the fungus. As sucrose responsiveness is the main determinant of division of foraging labour, these changes might unsettle the numerical ratio between the sub-castes of foragers leading to suboptimal foraging. Although the use of biocontrol strategies should be preferred over chemical pesticides, careful assessment of their side-effects is crucial before claiming that they are safe for pollinators.

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

ABSTRACTSearching 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.