Metal pollutants have additive negative effects on honey bee cognition

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.

Methods
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.

环境污染物可对动物产生亚致死性有害影响，其中包括破坏支撑关键行为的认知功能。尽管农药已被证实是传粉昆虫的主要威胁，但常以复合混合物形式存在的金属污染物迄今仍未受到足够重视。本研究评估了急性暴露于三种常见金属污染物——铅、铜、砷的田间现实浓度及其复合暴露时，对西方蜜蜂（*Apis mellifera* Linnaeus 1758）奖赏性学习与记忆的影响。单一金属处理组均延缓了学习进程，并破坏了24小时时的记忆提取能力；金属复合处理则对这两项过程产生了叠加式负面影响，提示存在共同的毒性通路。本研究结果凸显了进一步评估金属污染对无脊椎动物生存风险的必要性。

## 实验方法
### 蜜蜂金属暴露处理
本研究于2020年8月的清晨，在5个不同蜂箱的入口处收集归巢的采集蜂（西方蜜蜂*Apis mellifera* Linnaeus 1758）。将蜜蜂置于冰面麻醉后，固定于塑料试管中，并用胶带与头部后方的一滴石蜡固定（Matsumoto等，2012）。采用50%蔗糖溶液刺激蜜蜂触角，以此筛选出具有完整伸喙反射（Proboscis Extension Reflex, PER）的个体。随后为产生伸喙反射的蜜蜂饲喂5 μL 50%蔗糖溶液（金属暴露处理详见下文），确保其完全摄入该溶液，并将其置于恒温恒湿培养箱中静置3小时（温度：25±2℃，湿度：60%）。对蔗糖溶液无伸喙反射的蜜蜂将被剔除。

### 绝对学习训练
条件化训练前，再次采用50%蔗糖溶液刺激触角筛选具有完整伸喙反射的蜜蜂，仅保留显示该反射的个体。随后依据标准流程，使用自动化刺激递送系统开展嗅觉绝对条件化训练（Aguiar等，2018）。训练中，蜜蜂需学会对嗅觉条件刺激（Conditioned Stimulus, CS：1-壬醇，Sigma-Aldrich有限公司）产生响应，该刺激将由非条件刺激（Unconditioned Stimulus, US：50%蔗糖溶液）强化。训练共包含5轮条件化试验，每轮试验间隔10分钟。
每轮试验总时长为37秒：首先将蜜蜂置于刺激递送系统前方，系统持续向蜜蜂触角输送清洁空气（流速3300 mL·min⁻¹）；15秒后，将气味注入气流中，持续4秒，其中最后1秒与牙签蘸取蔗糖溶液触碰触角的时间重合；随即立即将牙签置于蜜蜂喙部，供其取食4秒；随后蜜蜂继续在清洁气流中停留15秒。每轮试验中，记录蜜蜂在气味刺激下是否产生条件化伸喙反射（记为1或0）。首次试验即自发产生响应的蜜蜂将被剔除出后续分析。所有试验中条件化响应的总次数即为个体的习得得分（取值范围为0~4），在最后一轮试验产生响应的蜜蜂被归类为“学习者”。

### 长期记忆测试
仅将完成学习任务的蜜蜂纳入记忆性能分析。训练结束后，为这些蜜蜂饲喂15 μL 50%蔗糖溶液，置于培养箱中过夜，并于次日清晨再次饲喂5 μL蔗糖溶液。训练后24小时（即静置3小时后）开展记忆保持测试：测试包含3轮与训练流程相似的试验，但不提供蔗糖奖励。除训练中使用的条件刺激气味（CS）外，还将以随机顺序呈现两种新型气味，以评估记忆的特异性：壬醛被认为与1-壬醇的气味感知相似，而1-己醇则被认为与前者气味感知不同（Guerrieri等，2005）。每轮记忆保持试验中，记录蜜蜂对每种气味是否产生条件化伸喙反射（记为1或0）。根据蜜蜂的响应模式将其分为四类：仅对CS产生响应、对CS与相似气味均产生响应（泛化水平较低）、对所有气味均产生响应（泛化水平较高），以及无响应或响应不一致。