FIGURE 1

Germ-free zebrafish colonized with GFP labeled Escherichia coli DH5α. (A) Fluorescence images of GF zebrafish and GF zebrafish mono-associated with 102, 104, 105, 106 CFU/ml E. coli DH5α at 3 dpf for 24 h. The magnification of the fluorescence microscope is 3.5×. (B) The survival rate of germ-free (GF) zebrafish, conventional raised (CONR) zebrafish and germ-free zebrafish exposed to different concentrations of E. coli DH5α at 3 dpf for 24 or 48 h. (C) The survival rate of germ-free (GF) zebrafish, conventionally raised (CONR) zebrafish and germ-free zebrafish exposed to different concentrations of E. coli DH5α at 5 dpf for 24 or 48 h. (D) Colonization concentrations (CFU/fish) of mono-associated zebrafish colonized at 3 dpf for 24 or 48 h. (E) Colonization concentrations (CFU/fish) of mono-associated zebrafish at 5 dpf for 24 or 48 h. All data (B–E) are presented as means ± SEM, n = 45 per group.
FIGURE 2

Germ-free zebrafish colonized with GFP labeled Bacillus subtilis WB800N. (A) Fluorescence images of germ-free (GF) zebrafish and germ-free (GF) zebrafish mono-associated with different concentrations of B. subtilis WB800N (102, 104, 105, 106CFU/ml). The magnification of the fluorescence microscope is 3.5×. (B) The survival rate of germ-free (GF) zebrafish, conventional raised (CONR) zebrafish and germ-free zebrafish exposed to different concentrations of B. subtilis WB800N at 3 dpf for 24 or 48 h. (C) The survival rate of GF zebrafish, conventionally raised (CONR) zebrafish and germ-free zebrafish exposed to different concentrations of B. subtilis WB800N at 5 dpf for 24 or 48 h. (D) Colonization concentrations (CFU/fish) of mono-association zebrafish colonized at 3 dpf for 24 or 48 h. (E) Colonization concentrations (CFU/fish) of GF zebrafish at 5 dpf for 24 or 48 h. All data (B–E) are presented as means ± SEM, n = 45 per group.
FIGURE 3

Comparisons of gene expression levels among germ-free (GF) zebrafish, CONR zebrafish and mono-associated (GF) zebrafish with different concentrations of E. coli DH5α. (A) The expression levels of nutrients metabolism related genes fiaf, (B)cpt1a, (C)gk.(D) The expression level of cell proliferation related genes, pcna, (E)state3, (F)cdc25a.(G) The expression levels of innate immune related genes il-10, (H)tnf-α and (I)il-1β. (J) The expression levels of damage stress related genes hsp70, (K)xbp1 and (L)atf6. The results were shown as mean ± SEM of three independent experiments with six replicates for each experiment, n = 90, per group. Different letters indicate statistically significant differences among groups (p < 0.05).
FIGURE 4

Comparisons of gene expression levels among germ-free (GF) zebrafish, CONR zebrafish and mono-associated (GF) zebrafish with different concentrations of B. subtilis WB800N. (A) The expression levels of nutrients metabolism related genes fiaf, (B)cpt1a, (C)gk. (D) The expression level of cell proliferation related genes pcna, (E)state3, (F)cdc25a. (G) The expression levels of innate immune related genes il-10, (H)tnf-α and (I)il-1β. (J) The expression levels of damage stress related genes hsp70, (K)xbp1 and (L)atf6. The results were shown as mean ± SEM of three independent experiments with six replicates for each experiment, n = 90, per group. Different letters indicate statistically significant differences among groups (p < 0.05).
FIGURE 5

Behavior detection of CONR larvae, germ-free (GF) larvae and germ-free larvae mono-associated with 104 and 106 CFU/ml E. coli DH5α. (A) Traveling distance was recorded every minute, testing for 48 h in total and traveling distance during 5 h when the behavior of all zebrafish was relatively stable is shown. (B) Mean traveling distance of each group during the 48 h of testing. (C) The distance that CONR), germ-free (GF) and germ-free (GF) mono-associated zebrafish moved in slow (<8 mm/s), middle (>8 and <20 mm/s) or fast (>20 mm/s) locomotion during 48 h of testing. (D) The expression levels of 5-hydroxytryptamine synthesis related genes htr1aa, tph1a, slc4a6a, mao, and bdnf. All data (B–D) were presented as means ± SEM. Different letters indicate statistically significant differences among groups (p < 0.05).
FIGURE 6

Behavior detection of CONR larvae, germ-free (GF) larvae and germ-free (GF) larvae mono-associated with 104 and 106CFU/ml B. subtilis WB800N. (A) Traveling distance was recorded every minute, testing for 48 h in total and traveling distance of a period of 5 h is shown. (B) Mean traveling distance of each group during the 48 h of testing. (C) The traveling distance that CONR larvae, germ-free (GF), and germ-free (GF) mono-associated zebrafish moved in slow (<8 mm/s), middle (>8 and <20 mm/s), or fast (>20 mm/s) locomotion during 48 h of testing. (D) The expression levels of 5-hydroxytryptamine synthesis related genes htr1aa, tph1a, slc4a6a, mao, and bdnf. All data (B–D) were presented as means ± SEM. Different letters indicate statistically significant differences among groups (p < 0.05).
Acknowledgments
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