Supplementary Methods from Fungicide suppression of flight performance in the honeybee (Apis mellifera) and its amelioration by quercetin

As a managed agricultural pollinator, the western honeybee Apis mellifera frequently encounters agrochemicals as contaminants of nectar and pollen. One such contaminant, the fungicide boscalid, is applied at bloom in orchards for fungal floral pathogen control. As an inhibitor of complex II in the mitochondrial electron transport chain of fungi, boscalid can potentially interfere with high energy-demanding activities of bees, including flight. We designed an indoor flight treadmill to evaluate impacts of ingesting boscalid and/or quercetin, a ubiquitous phytochemical in bee food that also affects mitochondrial respiration. Boscalid reduced the wingbeat frequencies of foragers during flight but did not alter the duration of flight. At the colony level, boscalid ingestion may thereby affect overall health by reducing forager efficiency. The consumption of quercetin, by contrast, led to higher adenosine triphosphate levels in flight muscles and a higher wingbeat frequency. Consuming the two compounds together increased wingbeat frequency, demonstrating a hitherto unrecognized mechanism by which dietary phytochemicals may act to ameliorate toxic effects of pesticides to promote honeybee health. In carrying out this work, we also introduce two methodological improvements for use in testing for pesticide effects on flight capacity—a ‘force-feeding’ to standardize flight fuel supply and a novel indoor flight treadmill.