Fig 2: Effects of experimental treatments on the gill cellular stress response in adult female zebrafish. (A) Gill hsf1, hsp70a, hsp90aa and hsp47 relative gene expression. (B) Representative western blot and HSP70, HSP90 and HSP47 relative protein expression. Gene expression data were normalized and expressed as stated in Fig. 2. Western blot shows HSP70 standard (lane 1), HSP90 standard (lane 2), pool of heat-stressed gills (positive control; lane 3), control treatment (lane 4), cycling temperature treatment (lane 5), cycling hypoxia treatment (lane 6), combined exposure treatment (lane 7) and blank (lane 8). Dashed lines around a lane represent the splicing of separate gel images. Protein expression was normalized to Coomassie stain band intensity and expressed relative to the control treatment for each protein. Values are means+s.e.m. (hsf1, n=7–8; hsp70a, hsp90aa and hsp47, n=6–7; HSP70, HSP90 and HSP47, n=5–6). Statistical differences between gene expression values were determined by Kruskal–Wallis one-way ANOVA followed by post hoc Dunn's test (hsf1, P<0.001; hsp70a, P=0.363; hsp90aa, P<0.001; hsp47, P<0.001). HSP70 protein expression was square-root transformed prior to analysis; statistical differences between protein expression values were determined by one-way ANOVA followed by post hoc Holm–Šidák tests (HSP70, P<0.001; HSP90, P=0.027) or a Kruskal–Wallis one-way ANOVA (HSP47, P=0.868). Values for a given parameter that do not share a common letter are significantly different from one another.

Fig 3: Effects of parental treatment on zebrafish embryo cellular stress response. (A) hsf1, hsp70a, hsp90aa and hsp47 relative gene expression, and (B) representative western blot and HSP70, HSP90 and HSP47 relative protein expression in ∼1 hpf embryos derived from adult zebrafish exposed to the experimental treatments. Gene expression data were normalized and expressed as stated in Fig. 2. Western blot bands and normalization of protein expression are as stated in Fig. 5. Values are means±s.e.m. (hsf1, hsp70a, hsp90aa and hsp47, n=5–6; HSP70, HSP90 and HSP47, n=5). Statistical differences between gene expression values were determined by Kruskal–Wallis one-way ANOVA followed by post hoc Dunn's test (hsf1, P=0.014; hsp70a, P<0.001; hsp90aa, P=0.002; hsp47, P<0.001). HSP70 protein expression was log-transformed prior to analysis; statistical differences between protein expression values were determined by one-way ANOVA followed by post hoc Holm–Šidák tests (HSP70, P=0.010; HSP47, P=0.175) or a Kruskal–Wallis one-way ANOVA followed by post hoc Dunn's test (HSP90, P=0.004). Values for a given parameter that do not share a common letter are different from one another.

Fig 4: Effects of experimental treatments on the ovary cellular stress response in adult female zebrafish. (A) Ovary hsf1, hsp70a, hsp90aa and hsp47 relative gene expression. (B) Representative western blot and HSP70, HSP90 and HSP47 relative protein expression. Gene expression data were normalized and expressed as stated in Fig. 2. Western blot bands and normalization of protein expression are as stated in Fig. 5. Values are means+s.e.m. (hsf1, n=9–10; hsp70a and hsp90aa, n=9–11; hsp47, n=7–9; HSP70, HSP90 and HSP47, n=5–6). Statistical differences between gene expression values were determined by Kruskal–Wallis one-way ANOVA followed by post hoc Dunn's test (hsf1, hsp70a, hsp90aa and hsp47, P<0.001). HSP90 protein expression was square-root transformed prior to analysis; statistical differences between protein expression values were determined by one-way ANOVA followed by post hoc Holm–Šidák tests (HSP90, P=0.288; HSP47, P=0.019) or a Kruskal–Wallis one-way ANOVA followed by post hoc Dunn's test (HSP70, P=0.002). Values for a given parameter that do not share a common letter are significantly different from one another.

Fig 5: Effects of parental treatment on zebrafish embryo cellular stress response. (A) hsf1, hsp70a, hsp90aa and hsp47 relative gene expression and (B) representative western blot and HSP70, HSP90 and HSP47 relative protein expression in ∼1 h post-fertilization (hpf) embryos derived from adult zebrafish exposed to either control or combined exposure conditions for 14 days. Gene expression data were normalized and expressed as stated in Fig. 2. Statistical differences between gene values were determined by two-tailed t-tests (hsf1: P<0.001, hsp90aa: P<0.001, hsp47: P=0.054) or a Mann–Whitney rank sum test (hsp70a: P=0.004). Representative western blot analysis of HSP70 standard (lane 1), HSP90 standard (lane 2), pool of heat stressed gills (positive control; lane 3), control treatment (lane 4), combined exposure treatment (lane 5) and blank (lane 6). Each western blot run contained multiple samples from each treatment group, but for the sake of presentation clarity, only a single replicate for each treatment is presented, requiring spliced images (represented by dashed lines). Protein expression was normalized to Coomassie stain band intensity and expressed relative to the control treatment for each protein. All statistical differences between protein values were determined by two-tailed t-tests (HSP70: P=0.046; HSP90: P<0.001; HSP47: P=0.374). Differences between treatments for a given parameter are indicated by an asterisk. Data are medians and interquartile ranges in A and are means±s.e.m. in B (hsf1, hsp70a, hsp90aa and hsp47, n=5–7; HSP70, HSP90 and HSP47, n=5–6).