Journal: bioRxiv

Article Title: Engineered symbiont biosensor maps micron-scale sugar gradients in the honeybee gut

doi: 10.1101/2025.11.28.691108

Figure Lengend Snippet: ( a ) Schematic outline of the in vivo experiment workflow. ( b ) Confocal microscopy images of a honeybee ileum co-colonized by S. alvi ac02 and G. apis ESL169 (left) or G. apicola ESL309 (right). The blue channel depicts DAPI staining of host and bacterial DNA. The green channel shows GFP fluorescence from the engineered S. alvi strain. Red channel shows E2-crimson fluorescence from the Gilliamella strains transformed with the pAC09 plasmid. Scale bar represents 100 μm. ( c ) G. apicola ESL309 catabolizes arabinose whereas G. apis ESL169 does not. Box plot shows median value of GFP fluorescence of S. alvi biofilms imaged from the gut of bees fed sugar water (-) or sugar water supplemented with 210 μM arabinose (ara). Each fluorescence value corresponds to the overall mean intensity averaged from 3 sections (anterior, mid-ileum, posterior) of one gut and across the depth of the bacterial biofilm for each section. Different letters indicate significant differences in fluorescence intensities across colonization and diet conditions, with adjusted p-value < 0.05 (Tukey HSD test). (d) Distribution of pollen-derived arabinose is longitudinally uniform along the ileum. Truncated violin plot shows median value of GFP fluorescence quantified from S. alvi biofilms imaged in the gut of bees fed sugar water (-) or sugar water and pollen ad libitum (pollen). Each fluorescence value corresponds to the mean intensity averaged across the depth of the bacterial biofilm for each gut section. (e) Arabinose derived from pollen breakdown distributes with high heterogeneity along a radial gradient across the depth of S. alvi ac02 biofilm when co-colonized with G. apis ESL169 (left) or G. apicola ESL309 (right). Graphs show the mean GFP fluorescence of the biosensor according to the biofilm depth of individual bees. Data corresponding to the anterior ileum region are displayed only for clarity. Complete data are provided in Supplementary Fig. 8.

Article Snippet: Gilliamella strains carried the plasmid pAC09 (Addgene plasmid No. 197403) encoding the E2-crimson fluorescent protein to enable in vivo visualization.

Techniques: In Vivo, Confocal Microscopy, Staining, Fluorescence, Transformation Assay, Plasmid Preparation, Derivative Assay