The previous state of colonization affects the ability of transposon-tagged libraries to colonize the zebrafish intestine. (A) Table showing the experimental situations where transposon-tagged libraries were exposed to zebrafish intestines at 6 days postfertilization (dpf) with different colonization states. Shapes used to represent different experimental conditions in all figures are shown, where solid squares represent WT bacterial populations and striped squares represent TAG libraries. (B and C) Abundance of unique TAGs detected among Aeromonas (B) and Vibrio (C) libraries in differentially colonized zebrafish. Each symbol represents the value for an individual zebrafish intestine, except for “inoculum,” which represents technical replicates of the same input libraries. The mean of each group of zebrafish is indicated by a short black line. Values that are significantly different are indicated by the bar and asterisk as follows: *, P < 0.01; **, P < 0.001. (D) The fluorescence intensity of two marked WT Aeromonas strains (HmTn7RFP [red] and HmTn7GFP [green]) is compared in nine fish where the second strain was given at 6 dpf to mimic the TAG experiments shown in panel B. The first marked strain allowed to colonize was swapped in four of the fish (so lines can connect red and green points or green and red points) to control for possible differences due to the incorporated fluorescent-protein-coding gene. Intensities have been scaled to reflect differences in brightness of the two colors. (E) Representative light sheet microscopy images of a zebrafish intestine colonized first by RFP-labeled bacteria (at 5 dpf) and then by GFP-labeled bacteria (at 6 dpf) and imaged at 7 dpf. This intestine had a particularly large amount of the second strain such that both can be seen well. D The intestine (inside the white dotted lines), vent (white arrows), and swim bladder (sb), which reflects fluorescence to some extent, are indicated.