Environmental pollutants can exert sublethal deleterious effects on animals. These include disruption of cognitive functions underlying crucial behaviours. While agrochemicals have been identified as a major threat to pollinators, metal pollutants, which are often found in complex mixtures, have so far been overlooked. Here we assessed the impact of acute exposure to field-realistic concentrations of three common metal pollutants, lead, copper, arsenic, and their combinations, on honey bee appetitive learning and memory. All treatments involving single metals slowed down learning and disrupted memory retrieval at 24 h. Combinations of these metals had additive negative effects on both processes, suggesting common pathways of toxicity. Our results highlight the need to further assess the risks of metal pollution on invertebrates.

Bees exposure to metals

We collected honey bees (Apis mellifera Linnaeus 1758) returning from foraging trips at the entrance of five different hives in mornings during August 2020. We anaesthetized the bees on ice and harnessed them in plastic tubes, secured with tape and a droplet of wax at the back of the head (Matsumoto et al., 2012). We tested all bees for an intact PER by stimulating their antennae with 50% sucrose. We then fed the responding honey bees 5 μl of 50% sucrose solution (see Metal exposure), making sure they consumed the whole droplet, and left them to rest for 3 h in the incubator (temperature: 25 ±2°C, humidity: 60%). Honey bees that did not respond to the sucrose solution were discarded.

Absolute learning

Prior to conditioning, we tested all honey bees for the PER by stimulating their antennae with 50% sucrose solution, and kept only those that displayed the reflex. We then performed olfactory absolute conditioning according to a standard protocol using an automatic stimulus delivery system (Aguiar et al., 2018). Honey bees had to learn to respond to an olfactory conditioned stimulus (CS, 1-nonanol; Sigma-Aldrich Ltd) reinforced with the unconditioned stimulus (US, 50% sucrose solution), over five conditioning trials with a ten-minute inter-trial interval. Each trial (37 s in total) began when a bee was placed in front of the stimulus delivery system, which released a continuous flow of clean air (3300 ml min−1) to the antennae. After 15 s, the odour was introduced into the airflow for 4 s, the last second of which overlapped with sucrose presentation to the antennae using a toothpick. This was immediately followed by feeding for 4 s by presenting the toothpick to the proboscis. The bee remained for another 15 s under the clean airflow. We recorded the presence or absence (1/0) of a conditioned PER in response to the odorant presentation during each conditioning trial. Honey bees spontaneously responding in the first conditioning trial were discarded from the analysis. The sum of conditioned responses over all trials provided an individual acquisition score (between 0 and 4), and honey bees responding at the last trial were categorized as learners.

Long-term memory

Only honey bees that had learnt the task were kept for the analysis of memory performance. After conditioning, these honey bees were fed 15 μl of 50% sucrose solution, left overnight in the incubator, and fed another 5 μl of sucrose solution the following morning. Three hours later (24h post-conditioning), we performed the retention test, consisting of three trials similar to conditioning except that no sucrose reward was presented. In addition to the odour used during the conditioning (CS), we presented two novel odours, in randomized order, to assess the specificity of the memory: nonanal was expected to be perceived by honey bees as similar to 1-nonanol, while 1-hexanol was expected to be perceived differently (Guerrieri et al., 2005). We recorded the presence or absence (1/0) of a conditioned PER to each odorant at each memory retention trial. We classified honey bees according to their response patterns: response to the CS only, response to the CS and the similar odour (low generalization level), response to all odours (high generalization level), no or inconsistent response.

See ReadMe file or tab 'ReadMe' of Dataset S1 excel file