Figure 4: DEICODE distances based on Robust Aitchison Principal Components Analysis. Points represent single samples colored according to treatment. Arrows represent Euclidian distances from the origin and indicate ASVs with strong influence on the principal component axis. Ellipses show 95% confidence interval for multivariate t-distribution of each treatment. The ASV of the eukaryotic organism Bombus rupestris can explained by a remaining non-specificity of the used primers (as analyzed by TestPrime, www.arb-silva.de).
Effect of pollutants on bumblebee gene expression
In the transcriptome analysis, we focused only on biologically relevant comparisons of treatments to prevent unnecessary inflation of reported results. We compared control vs. solvent control, control vs. DEP, control vs. brake dust, and flight control vs. DEP flight. The analysis for differently expresses genes (DEGs) revealed differences between our treatments. In total, 324 genes were differentially expressed in the DEP treatment compared to the control (low-count gene filter settings: CPM Filter=1, samples reaching CPM Filter=2). 165 genes were upregulated (LogFC > 1) and 159 genes downregulated (LogFC < -1), respectively (Figure A2 ). In the brake dust treatment only one gene was differentially expressed (upregulated) in comparison to the control. In the solvent control, there were no differentially expressed genes compared to the control. In the DEP flight treatment, we found no differentially expressed genes in comparison to the flight control.
The variation in gene expression of bumblebee workers is clearly distinct between the control and the diesel exhaust particle treatment (Figure 5 ). The clear separation between the treatments across all samples indicates substantial differences in gene expression of bumblebees when exposed to DEP orally. The reliability of this difference in gene expression is confirmed by a cluster analysis which shows a definite clustering by treatment rather than by colony (Figure 6 ). The other treatments are not clearly distinct in a nMDS plot and indicate no differences in gene expression (Figures A3-A5 ), thus we do not conduct further analyses on these comparisons.