Glyphosate (GLY) is a broad-spectrum herbicide used for weed control. The sub-lethal impact of GLY on non-target organisms such as insect pollinators has not yet been evaluated. Apis mellifera is the main pollinator in agricultural environments and is a well-known model for behavioural research. Honeybees are also accurate biosensors of environmental pollutants and their appetitive behavioural response is a suitable tool with which to test sub-lethal effects of agrochemicals. We studied the effects of field-realistic doses of GLY on honeybees exposed chronically or acutely to the herbicide. We focused on sucrose sensitivity, elemental and non-elemental associative olfactory conditioning of the proboscis extension response (PER), and foraging-related behaviour. We found a reduced sensitivity to sucrose and learning performance for the groups chronically exposed to GLY concentrations within the range of recommended doses. When olfactory PER conditioning was performed with sucrose reward with the same GLY concentrations (acute exposure), elemental learning and short-term memory retention decreased significantly compared with controls. Non-elemental associative learning was also impaired by an acute exposure to GLY traces. Altogether, these results imply that GLY at concentrations found in agro-ecosystems as a result of standard spraying can reduce sensitivity to nectar reward and impair associative learning in honeybees. However, no effect on foragingrelated behaviour was found. Therefore, we speculate that successful forager bees could become a source of constant inflow of nectar with GLY traces that could then be distributed among nestmates, stored in the hive and have long-term negative consequences on colony performance.

The honeybee Apis mellifera is an important pollinator in both undisturbed and agricultural ecosystems. Its great versatility as an experimental model makes it an excellent proxy to evaluate the environmental impact of agrochemicals using current methodologies and procedures in environmental toxicology. The increase in agrochemical use, including those that do not target insects directly, can have deleterious effects if carried out indiscriminately. This seems to be the case of the herbicide glyphosate (GLY), the most widely used agrochemical worldwide. Its presence in honey has been reported in samples obtained from different environments. Hence, to understand its current and potential risks for this pollinator it has become essential to not only study the effects on honeybee colonies located in agricultural settings, but also its effects under laboratory conditions. Subtle deleterious effects can be detected using experimental approaches. GLY negatively affects associative learning processes of foragers, cognitive and sensory abilities of young hive bees and promotes delays in brood development. An integrated approach that considers behavior, physiology, and development allows not only to determine the effects of this agrochemical on this eusocial insect from an experimental perspective, but also to infer putative effects in disturbed environments where it is omnipresent.

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