DOI: 10.5061/dryad.ghx3ffbms
title: Metal pollutants have additive negative effects on honey bee cognition

description: 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.
description: <strong>Bees exposure to metals</strong>
 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.
 <strong>Absolute learning</strong>
 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.
 <strong>Long-term memory</strong>
 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.
description: See ReadMe file or tab 'ReadMe' of Dataset S1 excel
 file