Pollen Elicits Proboscis Extension but Does not Reinforce PER Learning in Honeybees

Abstract

The function of pollen as a reward for foraging bees is little understood, though there is evidence to suggest that it can reinforce associations with visual and olfactory floral cues. Foraging bees do not feed on pollen, thus one could argue that it cannot serve as an appetitive reinforcer in the same way as sucrose. However, ingestion is not a critical parameter for sucrose reinforcement, since olfactory proboscis extension (PER) learning can be conditioned through antennal stimulation only. During pollen collection, the antennae and mouthparts come into contact with pollen, thus it is possible that pollen reinforces associative learning through similar gustatory pathways as sucrose. Here pollen was presented as the unconditioned stimulus (US), either in its natural state or in a 30% pollen-water solution, and was found to elicit proboscis extension following antennal stimulation. Control groups were exposed to either sucrose or a clean sponge as the US, or an unpaired presentation of the conditioned stimulus (CS) and pollen US. Despite steady levels of responding to the US, bees did not learn to associate a neutral odour with the delivery of a pollen reward, thus whilst pollen has a proboscis extension releasing function, it does not reinforce olfactory PER learning.

Keywords: PER; conditioning; honeybee; learning; pollen; reinforcement.

Figure 1

(a) Acquisition curves and (b) PER response to US stimulation of bees rewarded at the antennae only with 30% sucrose solution (Black squares, n = 31), dry pollen (Black triangles, n = 31) or a clean sponge (Open diamonds, n = 29). Bars represent the number of bees responding to the conditioned stimulus (CS) in the final, unrewarded test (Black = Sucrose, White = Control, Grey = Pollen). Dashed line represents the overall spontaneous response to the CS on the first trial for all bees tested. Asterisks denote significant differences between treatment groups (** p <0.001 * p <0.05).

Figure 2

Proportion of bees responding to the CS (a) or US (b) on each trial. The US was either 30% sucrose solution (Squares) or a clean sponge (Circles). Bees were either exposed to an open dish of pollen during conditioning (Open shapes, n = 53) or a closed dish (Black shapes, n = 48). Bars represent the number of bees responding to the CS in the unrewarded tests. (Black bars=Sucrose, Grey bars=Control, Striped=Exposed to pollen; Striped= Non-exposed). In Test 1 olfactory conditions were matched to those experienced during training and in Test 2 the pollen dish was covered for all bees. The dashed line represents the overall spontaneous response to the CS on the first trial for all bees tested. Asterisks denote significant differences between treatment groups (** p < 0.001 * p < 0.05).

Figure 3

Proportion of bees extending the proboscis to antennal stimulation with a series of pollen-water mixtures, tested in order of ascending pollen concentration. Bees were separated according to forager type (Black circles = Pollen foragers, n = 53 Open circles = Non-pollen foragers, n = 55). Bars represent the response to a final stimulation with 30% sucrose (White = pollen foragers, Black = non-pollen foragers). The dashed lines represent bees’ responses to water stimulation prior to the stimulation with each pollen concentration (Black triangles = pollen foragers, Open triangles = non-pollen foragers).

Figure 4

Proportion of bees extending the proboscis to a previously neutral odour following pairing with different unconditioned stimuli. For trials 1–3, the US was either 30% pollen (w/w) solution (Black triangles, forward paired, n = 34, White diamonds, backward paired, n = 34) or 30% sucrose (w/w) solution (Black squares, n = 32). All bees received 30% sucrose on trials 4–6. The US was applied to both the antennae and proboscis.