Associative learning during early adulthood enhances later memory retention in honeybees

Abstract

Background: Cognitive experiences during the early stages of life play an important role in shaping the future behavior in mammals but also in insects, in which precocious learning can directly modify behaviors later in life depending on both the timing and the rearing environment. However, whether olfactory associative learning acquired early in the adult stage of insects affect memorizing of new learning events has not been studied yet.

Methodology: Groups of adult honeybee workers that experienced an odor paired with a sucrose solution 5 to 8 days or 9 to 12 days after emergence were previously exposed to (i) a rewarded experience through the offering of scented food, or (ii) a non-rewarded experience with a pure volatile compound in the rearing environment.

Principal findings: Early rewarded experiences (either at 1-4 or 5-8 days of adult age) enhanced retention performance in 9-12-day-conditioned bees when they were tested at 17 days of age. The highest retention levels at this age, which could not be improved with prior rewarded experiences, were found for memories established at 5-8 days of adult age. Associative memories acquired at 9-12 days of age showed a weak effect on retention for some pure pre-exposed volatile compounds; whereas the sole exposure of an odor at any younger age did not promote long-term effects on learning performance.

Conclusions: The associative learning events that occurred a few days after adult emergence improved memorizing in middle-aged bees. In addition, both the timing and the nature of early sensory inputs interact to enhance retention of new learning events acquired later in life, an important matter in the social life of honeybees.

Figure 1. Schematic schedule of the experimental series along the adult lifespan of the honeybee.

(a) Caged bees were offered a scented sugar solution for four consecutive days (gray boxes), and their olfactory memories at 17 days of age were evaluated in the PER paradigm (black arrow). (b) In addition to the scented solution received in a), bees were offered an alternative scented food for four consecutive days (1–4 or 5–8 days old, dark gray boxes). Then, bees were stimulated at: 1–4 + 5–8, 1–4 + 9–12 and 5–8 + 9–12 days of age. (c) An odor was exposed as volatile compound for four consecutive days (crossed boxes) before caged bees were offered the scented sugar solution (gray boxes). Odor exposure memory was not tested. (d) Caged bees were exposed to a pure odor for four consecutive days (crossed boxes) before conditioning (gray arrow). Notice that a non-odor exposed group was also included.

Figure 2. PER of early olfactory memories.

Long-term memories were quantified during five testing events in the proboscis extension response (PER) assay in 17-day-old bees that were either offered scented sugar solutions or exposed to odors during an early period of their adult lifespan. LIO (left panel) or PHE (right panel) were used as the tested odors when LIO (a) or PHE (b) were offered alone in the sugar solution (circles); when PHE (c) or LIO (d) were the non-tested odors used to scent the alternative aromatized food (triangles); and when PHE (e) or LIO (f) were the non-tested odors used as volatiles exposed in the rearing environment (squares). Number of subjects is indicated in brackets at the legend.

Figure 3. Differential conditioning after early odor exposure.

The learning performance was quantified during four conditioning trials in the proboscis extension response (PER) assay in 17-day-old bees that were exposed to a single odor as volatile in the rearing environment during different age periods: 1–4 days of age (circles); 5–8 days of age (squares), 9–12 days of age (triangles) and controls (rhombuses); We used closed symbols for CS+ and open ones for CS−. LIO as the rewarded odor (CS+) and PHE as the non-rewarded odor (CS−) were used in the differential conditioning when LIO (a) or PHE (b) were early exposed. PHE as the rewarded odor (CS+) and LIO as the non-rewarded odor (CS−) were used in the differential PER conditioning when LIO (c) or PHE (d) were early exposed. Number of subjects is indicated in brackets at the legend.