With both managed and wild bees facing many stressors6, it is important to fully understand the mechanisms driving performance impairments at the individual and colony levels. Unpacking the “black box” of sub-lethal effects on behaviour is essential for designing appropriate screening assays, and for implementing appropriate mitigation strategies52. Indeed, given regulatory changes in the use of neonicotinoid pesticides in some parts of the world, now is a critical time to be asking whether current approaches are optimal for estimating the magnitude of possible sub-lethal effects from alternative or newly developed pesticides bees may soon face72. Studies addressing the chronic, field-realistic exposure to any pesticide are a gold standard for estimating overall effects on colony foraging performance and success15,16. However, if we are to understand exactly how a pesticide affects bee behaviour and cognition, we need detailed behavioural experiments in controlled settings, paired with studies addressing effects at the neural level25. We asked if and how a neonicotinoid pesticide affects free-flying bees’ ability to learn about floral features, which allowed us to consider how acute exposure might impact not only the ability to learn, but also other foraging-related behaviour (Fig. 2). As a whole, our experiments demonstrated that a single acute dose of imidacloprid disrupted many foraging-related behaviours, but did not impair bees’ ability to learn an association between floral colour and a sucrose reward.
After ingesting a single dose of imidacloprid, one of the primary effects on behaviour was a reduction in bees’ apparent motivation to forage: in the higher-dose treatments, bees were less likely to visit flowers, collected less sucrose and were less likely to return to forage once having returned to the colony. Other studies have previously found that neonicotinoids suppress feeding motivation in bumblebees48,49,73,74,75. Free-flying studies have also found that neonicotinoids might cause a decrease in bumblebees’ tendency to forage for nectar (76 but see14); other studies have found that neonicotinoids reduce pollen foraging (14,15,16,17,77 but see78). Similarly, honeybees exposed to acute levels of imidacloprid were less likely to initiate foraging and made fewer foraging trips (79, see also80,81,82).
Another clear behavioural effect of imidacloprid was that, after landing on a flower, dosed bees were more likely to run over it without stopping to visit the sucrose well. This particular impairment persisted through both trials in Experiment 2 (some 90 minutes following pesticide exposure). Bypassing the nectar in this way may have been due to hyperactivity or impairment to motor coordination83,84. In line with previous findings in honeybees83,85, it seemed that in our study more broadly that bumblebees were initially hyperactive, followed by hypoactive. Indeed, bees in higher-dose treatments that did not land were often seen to fly or run around flowers for 5–10 minutes, followed by crawling into a corner of the arena and remaining motionless. Once back in the colony, individuals appeared to be less active: instead of moving around the colony in “excited runs”70 as newly returning foragers often do71, they would instead be stationary, or crawl underneath the colony. Interestingly, in Experiment 2, when we manually returned these foragers to the foraging arena for their second learning trial or test, these bees would generally commence foraging. Effects on activity levels or motor coordination could be detrimental both to bees’ ability to fly85 as well as to their ability to handle natural, morphologically complex flowers. Indeed, bumblebees exposed to chronic levels of thiamethoxam visited individual wildflowers flowers more times before gaining a nectar reward from them78, which could be evidence of motor deficits.
Despite the clear dose-dependent negative effects we found on a number of foraging-related behaviours, we did not find impairment to bees’ ability to learn which flowers were rewarding based on colour. This implies that at least in this scenario, foraging deficits are more likely to be due to the motivational and motor impairments we found, rather than the ability to learn. While a few studies have addressed acute effects of neonicotinoids on honeybee learning35,36,37, and a few have addressed the effects of chronic exposure to bumblebees40,41,76,86, we only found one study that addressed acute effects to bumblebee learning40. In that experiment, individual harnessed bumblebees were fed 10 μl of control, 2.4, 10 or 250 ppb thiamethoxam before being tested on a learning task an hour later. Bees in the two highest dose treatment groups had a lower “learning level” (the total number of times they extended their proboscis in response to the conditioned odour stimulus), and bees in the highest treatment group were less “trainable” (whether they learned the association or not over the 15 trials). There are a number of factors that differ between that study and our own, but we believe that the two most probable explanations of the difference in results are the conditioned stimuli the bees were trained to (olfactory versus visual) and the learning protocol (harnessed versus free-moving). These two features dominate studies of neonicotinoid effects on learning more broadly (Fig. 1; Table S1), and so they might also explain the difference in results in our study and the more general trend of results that find that neonicotinoids impair learning18.
With regard to differences in the modality of the conditioned stimulus, at least in honeybees, olfactory coding is impaired by a neonicotinoid25. This raises the possibility that bees may be less responsive to an olfactory stimulus because of difficulty in olfactory discrimination; if so, learning in other modalities may not be equivalently impaired. In order to determine this, one would need to carry out the same protocol but with conditioned stimuli in multiple modalities (e.g. visual vs. olfactory).
With regard to the second main difference in learning protocol, in our study bees were free-flying, as opposed to being immobilized in harnesses for PER. One limitation of the PER protocol is that it can often be difficult to differentiate between impairment to feeding/foraging motivation and learning performance, since both result in the same behavioural output: no proboscis extension to the trained stimulus. Researchers often attempt to control for this ambiguity by separately accounting for the effects of pesticide exposure on sucrose responsiveness; some have found that neonicotinoids reduce sucrose responsiveness35,38,62, while others have found no effect37,40,41. Even by excluding individuals that are less responsive to sucrose (as defined by some response threshold), there may still be motivational differences between treatment groups. For example, in our study, most of the bees we tested drank on the first (white) artificial flowers when we stimulated their antennae with sucrose, even though many of these bees did not then subsequently visit flowers. If this experiment had been in a PER setup, it is probable that many of these bees would have been included, since they would have been responsive to sucrose, but may have not been motivated enough to respond to an odour alone. In contrast to PER, in free-flying studies, behaviours associated with foraging motivation (i.e. propensity to visit flowers, amount of nectar collected) are different to the behaviour measured for learning performance (correct vs. incorrect floral choices), and thus motivation vs. learning performance can be more straightforwardly differentiated.
While the vast majority of studies investigating neonicotinoid effects on learning in bees have been in relation to olfactory stimuli (Fig. 1), there are three studies addressing effects on free-flying bees visiting visual stimuli76,79, which all have results compatible with our own, and bolster our finding that imidacloprid affects motivation more so than visual learning. In a recent study, B. terrestris chronically exposed to 1 ppb of imidacloprid were impaired in measures indicative of foraging motivation (time taken for bees to visit flowers and the number of flowers visited) but they were not impaired in their ability to learn which colour of flowers were rewarding86. In another recent study with B. impatiens, colonies that were chronically exposed to imidacloprid at 2.6 and 10 ppb were impaired in the number of trials that it took for individuals to develop preferences for the most highly rewarding flower (yellow) over three other less rewarding options76. While the reason behind this finding is not clear, the authors suggest it may be that treated bees were less motivated to forage (supported by the finding that they spent less time foraging and visited fewer flowers), and thus had less opportunity to learn. Finally, in a study with honeybees A. mellifera, individuals exposed to acute levels of imidacloprid were not impaired in their ability to learn that blue flowers were more highly rewarding than white, or in their ability to reverse this learned association79. However, interpretation of these results is difficult given that preference for blue flowers was affected by pesticide exposure when both flowers were equally rewarding.
Our study is a snapshot of the behavioural effects of a single dose of a neonicotinoid on an individual bee’s behaviour in the context of learning about colour, and of course many other factors could affect whether a neonicotinoid affects learning, such as the amount of time that a bee is exposed to a pesticide, the duration of time after exposure that we measure behaviour, as well as which neonicotinoid is used87. Even within a given pesticide treatment regime, all the factors known to affect learning, such as the salience of conditioned stimuli and motivational value of rewards88, might also affect our ability to detect pesticide effects on learning ability. In our experiment we chose stimuli that would be relatively difficult for bees to discriminate, to both be ecologically realistic (in natural foraging scenarios bees often need to discriminate between stimuli this similar in colour e.g.89), and to maximize the chances that we would detect an effect on learning if it existed in this context. We also replicated the experiment across two different scenarios where the motivational value of the US changed, as well as the opportunity to learn. These are factors that need to be considered in future studies if we are to determine not only if learning is impaired by a pesticide, but also whether such impairment has relevance for natural foraging scenarios, and thus what role such impairment might play relative to other effects on bee behaviour.
More broadly, our study highlights some of the factors that need to be considered when addressing the effects of a stressor on cognition. Specifically, we need to address cognition across more than a single (usually olfactory) modality, including consideration of how more realistic multi-modal floral stimuli90 might help compensate for foraging performance deficits previously documented in single-modality scenarios. We also need to address a broader range of cognitive abilities and learning scenarios. Given that bees use a wide range of cognitive abilities when foraging, from spatial cognition64 to learning non-elemental (configural) associations27,91, by focusing just on olfactory associative learning we are likely only seeing a part of the picture. In addition, a single protocol can only ever tell us about animal behaviour under certain conditions, and so a broader range of behavioural protocols, especially ones that address cognition under more ecologically realistic scenarios will be informative.