Consumption of pollen contaminated with field-realistic concentrations of fungicide cause sub-lethal effects in common eastern bumble bee (Bombus impatiens [Hymenoptera]: [Apidae]) microcolonies

Bumble bees are declining across the globe. Causes of this decline have been attributed to a variety of stressors, including pesticides. Fungicides are a type of pesticide that have been understudied in the context of bumble bee health. As a result, fungicides are often applied to flowering plants without consideration of pollinator exposure. Recent work demonstrates that fungicides have sublethal effects in bumble bees, but little is known about how much fungicide it takes to cause these sublethal effects. To address this gap in the literature, we fed microcolonies of the common eastern bumble bee (Bombus impatiens) pollen contaminated with a range of fungicide concentrations. We chose these concentrations based on the range of fungicide concentrations in pollen and nectar that were reported in the literature. Results revealed that later-stage pupae and newly emerged males are potentially sensitive to fungicide exposure, showing smaller size and reduced fat reserves at intermediate levels of contamination. Compared to the control, intermediated levels of fungicide-contaminated pollen led to increased pupal mortality and delayed male emergence. Contrary to expectations, higher fungicide levels did not exhibit a linear relationship with negative impacts, suggesting nuanced effects. Because body size and emergence timing are important aspects of bumble bee reproductive behavior, results have implications for mating success, potentially disrupting colony development.

Methods
To understand how fungicides affect bumble bee microcolony health and reproduction, we established five fungicide treatments, which varied by the concentration of Pristine® in their pollen diet. We selected experimental values of Pristine® of 0 ppb, 150 ppb, 1,500 ppb, 15,000 ppb and 150,000 ppb.We exposed bumble bees to fungicide by incorporating it into pollen balls, which we made in batches per treatment by mixing 90g of honey bee-collected pollen with 35ml of a 50/50 sucrose:water solution containing the appropriate concentration of fungicide. We made pollen balls for each treatment in separate batches, from a single supply of homogenized honey bee-collected pollen. Using callow worker bees from four purchased B. impatiens queen-right colonies (Plant Products USA, Westland, MI), we established microcolonies consisting of five individuals, supplied with a 50/50 sucrose:water solution, a 1 g (±0.5 g) fungicide-free pollen ball, and 1-2 g of wax material from their natal colony to stimulate oviposition and brood production behavior. We replicated each of the five fungicide treatments nine times, for a total of 45 microcolonies. We placed microcolonies in a rearing room under red light at The Ohio State University’s Rothenbuhler Honey Bee Research Laboratory. Rearing room conditions were kept between 30°C-32°C, and 50%-70% relative humidity. We blocked by location within the rearing room, where each block contained microcolonies from each fungicide treatment and ensured that all workers within a block originated from the same original queenright colony to control for potential confounding genetic differences. Our experiment ran from August to October 2022, during which time we monitored the pollen consumption, brood production, and mortality in the 45 microcolonies. We stored data in Microsoft Excel, and ran all analyses in R.