Olfactory Learning in the Stingless Bee Melipona eburnea Friese (Apidae: Meliponini)

Abstract

Olfactory learning and floral scents are co-adaptive traits in the plant-pollinator relationship. However, how scent relates to cognition and learning in the diverse group of Neotropical stingless bees is largely unknown. Here we evaluated the ability of Melipona eburnea to be conditioned to scent using the proboscis extension reflex (PER) protocol. Stingless bees did not show PER while harnessed but were able to be PER conditioned to scent when free-to-move in a mini-cage (fmPER). We evaluated the effect of: 1) unconditioned stimulus (US) reward, and 2) previous scent-reward associations on olfactory learning performance. When using unscented-US, PER-responses were low on day 1, but using scented-US reward the olfactory PER-response increased on day 1. On day 2 PER performance greatly increased in bees that previously had experienced the same odor and reward combination, while bees that experienced a different odor on day 2 showed poor olfactory learning. Bees showed higher olfactory PER conditioning to guava than to mango odor. The effect of the unconditioned stimulus reward was not a significant factor in the model on day 2. This indicates that olfactory learning performance can increase via either taste receptors or accumulated experience with the same odor. Our results have application in agriculture and pollination ecology.

Keywords: Free-moving-PER (fmPER); conditioning protocols; learning; olfactory conditioning, cognitive ecology; pollinators; scented-US; stingless-bees.

Figure 1

Apparatus used in scent conditioning in the stingless bee Melipona eburnea using the proboscis extension reflex (PER) with free-moving bees (fmPER). A bee housed in a glass vial in which the cap is replaced with plastic mesh. The bee is exposed to the trained scent (conditioned stimulus (CS)), and a plume of scented airflow is dispensed with a syringe. The unconditioned response is elicited by touching the antenna with the unconditioned stimulus (US). Conditioned PER occurs when the bee extends its proboscis through the mesh in response to the CS presented before receiving the US. The US was provided with the tip of a toothpick.

Figure 2

Average US PER response (±SE) in all eight experiments for day 1 (light bars) and day 2 (dark bars) using caged stingless bees, Melipona eburnea.

Figure 3

Probability of the conditioned response PER in the stingless bees, Melipona eburnea on day 1. Bees conditioned with scented-US reward increased the conditioned response. The increment occurred along the trials, being higher when the conditioned odor was guava (G) than when it was mango (M) (model: Conditioned Response ~ Trials + Reward + Odor + (1 | Bee_Total)). The 95% confidence intervals are shown.

Figure 4

Probability of the conditioned response PER in the stingless bees, Melipona eburnean, on day 2. Bees that experienced the same odor the day before increased the conditioned response. The increment occurred along the trials, being higher when the conditioned odor was guava (G) than when it was mango (M) (model: Conditioned Response ~ Trials + Experience + Odor + (1 | Bee_Total) + Trials: Experience). The 95% confidence intervals are shown.