## 3 Results
### E. coli Associates with Adult D. melanogaster and Locates Preferentially in the Crop
#### 3.2.1 Bacterial Load of _E. coli_ Associated with _D. melanogaster_ Increases over Time  
To avoid any impact of the residing endogenous microbiome on my results, I associated starved, axenic adult males by feeding them briefly with an inoculum of an _E. coli_ lab strain, resistant to KM and containing _venus_ that encodes yellow fluorescent protein, which was deposited on fly food. Next, I transferred the flies back to sterile solid fly food and incubated them for another week (see Section 2). At this point, flies showed variable bacterial loads, containing an average of \(\pm\)1500 CFUs with up to \(\pm\)32,000 CFUs per fly (Figure 2A), which were confirmed to be the applied _E. coli_ lab strain via their kanamycin resistance and fluorescence. Associations with _E. coli_ do not impact feeding, as gnotobiotic flies show feeding dynamics in the CAFE assay that are highly similar to the previously observed feeding trends of axenic flies (Figure 1B) and to the feeding pattern of a group of axenic flies of the same age (Figure S2A). At the end of the CAFE assay, the bacterial load significantly increased, reaching an average of \(\pm\)30,000 CFUs per fly (Figure 2A). This increase is independent of the CAFE setup, as flies of the same age, maintained on the regular solid food source, showed a similar increase (Figure 2A). The much lower survivorship of _E. coli_-associated flies in the CAFE setup compared to previous axenic groups in the same assay (Figure S2B and Figure 1B), while surprising, should not be considered a confounding effect in the context of the increased bacterial load as care was taken to only sample living flies. One could argue that the increase in bacterial load indicates virulence and pathogenicity. However, the used strain is not known to be pathogenic, and axenic flies of the same age show similar increased mortality when maintained in the CAFE setup (Figure S2B). Furthermore, _E. coli_-associated or axenic flies of the same age, maintained on solid food, do not show any increased mortality (Figure S2B). Thus, not the association with _E. coli_, the CAFE setup nor the age of flies alone can explain the increased mortality. Instead, an interaction between fly age and the CAFE setup seems to lie at the basis of the increased mortality, and altogether, _E. coli_ seems to associate with _D. melanogaster_ in a quite stable fashion. Figure 2: _E. coli_ associates with the gut of _D. melanogaster_ when presented orally to axenic flies. (**A**) Bacterial load of _E. coli_ in the gut of _D. melanogaster_ was measured upon transferring gnotobiotic males (9–10 dpe) to a CAFE setup (full bars) or when flies were maintained on solid food (hatched bar at 96 h). Associations were obtained by feeding axenic, starved male flies (2–3 dpe) with an _E. coli_ inoculum and a subsequent incubation for another week on sterile solid fly food (see Section 2). Timepoint 0 h is before flies were transferred to the CAFE setup. Bacterial loads are normalized per fly (means \(\pm\) stdevs with \(n\geq\) 8 vials as gray points across \(\geq\)2 independent runs) and an lme with post hoc test showed a significant increase to the start at 96 h (Dunnett test above bars with *****, \(p\) < 0.0001). (**B**) The presence of _E. coli_ in the gut of _D. melanogaster_ was confirmed using microscopy on dissected guts. Widefield images (left) and a composite (right) of DAPI (staining the _D. melanogaster_ nuclei) and GFP (labeling _E. coli_ cells) images of one representative gut at 9–10 dpe (a second one is shown in Figure S3C). The hallmark structures of the crop (i), cardia (ii), copper cell region (iii), Malpighian tubules (iv) and rectal ampulla (v) are highlighted across the 3 main subdivisions of the fly gut. Whereas _E. coli_ cells can be observed in any segment of the gut of gnotobiotic flies, high bacterial densities are specifically found in (**C**) the crop and occasionally in (**D**) other gut regions when associated with the transit of food boluses of which some regions marked with a red square in (**B**) are magnified.