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Fig. 2 FIGURE 2. Heat-killed E. coli up-regulates p65 and zp2 and leads to a female-biased population. zp2 expression was determined by qRT-PCR from individuals (16, 19, and 21 dpf) using ef1a and 18 S rRNA as reference genes (A). Zebrafish juveniles were exposed to different concentrations of heat-killed bacteria, and zp2 expression was determined by qRT-PCR from individuals exposed from 15–21 dpf (B). Zebrafish juveniles at 20 and 41 dpf were exposed to heat-killed bacteria (107 cfu/ml) for 24 h. Analysis of p65 (C), sox9a (D), and amh (E) expression at 21 dpf juveniles and of p65 (F) expression at 42 dpf gonads was performed by qRT-PCR. Juveniles at 15 dpf were exposed to heat-killed E. coli for 20 days, and their sex was determined at 70 dpf. The mean and S.D. of three independent experiments is shown. Each group contained at least 20 individuals (G). Statistically significant differences between groups were determined using Student's t test for two groups and one-way analysis of variance test for more than two groups (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Error bars represent the mean ± S.D.