Floral nectar is a pivotal element of the intimate relationship between plants and pollinators. Nectars are composed of a plethora of nutritionally valuable compounds but also hundreds of secondary metabolites (SMs) whose function remains elusive. Here we performed a set of behavioural experiments to study whether five ubiquitous nectar non-protein amino acids (NPAAs: β-alanine, GABA, citrulline, ornithine and taurine) interact with gustation, feeding preference, and learning and memory in Apis mellifera. We showed that foragers were unable to discriminate NPAAs from water when only accessing antennal chemo-tactile information and that freely moving bees did not exhibit innate feeding preferences for NPAAs. Also, NPAAs did not alter food consumption or longevity in caged bees over 10 days. Taken together our data suggest that natural concentrations of NPAAs did not alter nectar palatability to bees. Olfactory conditioning assays showed that honey bees were more likely to learn a scent when it signalled a sucrose reward containing either β-alanine or GABA, and that GABA enhanced specific memory retention. Conversely, when ingested two hours prior to conditioning, GABA, β-alanine, and taurine weakened bees’ acquisition performances but not specific memory retention, which was enhanced in the case of β-alanine and taurine. Neither citrulline nor ornithine affected learning and memory. NPAAs in nectars may represent a cooperative strategy adopted by plants to attract beneficial pollinators.

Recent studies have reported that the ability to discriminate among quantities is not a prerogative of vertebrates. Ants, bees, and spiders can solve tasks in which they are required to discriminate between groups of objects. Although many studies regarding numerical cognition on invertebrates proposed a proto‐counting system, more control experiments for non‐numerical variables are necessary to confirm this hypothesis. Here, we developed a new method to investigate quantity discrimination abilities in invertebrates. We investigated the spontaneous choice of a cricket, Acheta domesticus. We exploited its natural shelter‐seeking behavior by presenting sets of geometrical shapes that simulated potential shelters. In a dichotomous choice between sets of geometrical black shapes differing in number of items, the majority of crickets chose the set containing the larger numerosity up to 2 versus 3 items. Control experiments suggested that crickets discriminated between sets consisting of different numbers of items by attending to continuous variables (i.e., convex hull and cumulative surface area) rather than by attending to numerosity. Secondly, when discriminating between single geometrical shapes, crickets attend to the width but not to the height of the stimuli.

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.

Floral nectar is a pivotal element of the intimate relationship between plants and pollinators and its chemical composition is likely to have been shaped by strong selective pressures. Nectars are composed of a plethora of nutritionally valuable compounds but also hundreds of secondary metabolites (SMs) whose ecological role is still not completely understood. Here we performed a set of behavioural experiments to study whether five ubiquitous nectar non-protein amino acids (NPAAs: β-alanine, GABA, citrulline, ornithine and taurine) interact with gustation, feeding preference, and learning and memory in the pollinator Apis mellifera. We showed that harnessed foragers were unable to discriminate NPAAs from water when only accessing antennal chemo-tactile information and that freely moving bees did not exhibit innate feeding preferences for NPAA-laced sucrose solutions. Also, dietary consumption of NPAAs did not alter food consumption or longevity in caged bees over 10 days. Taken together our data suggest that ecologically relevant concentrations of NPAAs did not alter nectar palatability to bees. Olfactory conditioning assays showed that honey bees were more likely to learn a scent when it signalled a sucrose reward containing either β-alanine or GABA, and that GABA also enhanced specific memory retention. Conversely, when ingested two hours prior to conditioning, GABA, β-alanine, and taurine weakened bees’ acquisition performances but not specific memory retention, which was enhanced in the case of β-alanine and taurine. Neither citrulline nor ornithine affected learning and memory. Our study suggests that NPAAs in nectars may represent a cooperative strategy adopted by plants to attract beneficial pollinators, while simultaneously enhancing pollen transfer among conspecific flowers. Future work should validate these results in more ecological scenarios and extend the study to as many nectar SMs as possible, alone and in combination, as well as to other species of pollinators.