Survivorship and gut microbial community of Bombus vosnesenskii exposed to an herbicide, a fungicide, an insecticide, or the combination of all three chemicals

We investigated site-level differences in survival and microbiome structure of wild bumble bees exposed to multiple pesticides, both individually and in combination. We collected wild Bombus vosnesenskii foragers (N = 175) from an alpine meadow, a valley lake shoreline, and a suburban park and maintained them on a diet containing an herbicide (glyphosate), a fungicide (tebuconazole), an insecticide (imidacloprid), or a combination of these chemicals. The herbicide treatment was 30 ppm of glyphosate (Sigma-Aldrich, USA), the fungicide treatment was 30 ppb of tebuconazole (Sigma-Aldrich, USA), and the insecticide treatment was 30 ppb of imidacloprid (Sigma-Aldrich, USA). These chemicals were selected because they had the highest estimated use for each chemical class (insecticide, herbicide, fungicide) in our region (Washoe county, Nevada, (Wieben 2019)). These concentrations of imidacloprid and tebuconazole approximate the median of those detected in nectar (Zioga et al. 2020). The concentration we chose for glyphosate is within the range of field-realistic exposure from treated plants (Herbert et al. 2014, Thompson et al. 2022, Helander et al. 2023b) although is likely higher than bees would experience foraging from non-target plants (Zioga et al. 2023). We monitored survival daily for 20 days. Every 3 days, we replaced feeders with a clean feeder with 1000 mL of solution. We continued the experiment until bees died or at day 20 (whichever came first) at which point we weighed the bees, measured their intertegular (IT) span to estimate body size (Cane 1987) and dissected out the midgut and hindgut (hereafter ‘gut’) for DNA extractions and 16s sequencing (see below).