Fig 2: 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 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 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).

Fig 4: Effects of parental treatment on zebrafish larvae cellular stress protein expression. Control and post-exposure to combined elevated temperature and hypoxia representative western blots and relative protein expression for (A) HSP70, (B) HSP90 and (C) HSP47 in 5 days post-fertilization (dpf) larvae derived from adult zebrafish exposed to either control (offspring control) or combined exposure conditions (offspring combined exposure) for 14 days. Western blot bands and protein expression was normalized and expressed as stated in Fig. 5. All protein expression was further normalized to control larvae derived from control parents. Statistical differences between values were determined by two-way ANOVA followed by a Holm–Šidák post hoc test (A: parental exposure: P=0.948; larval exposure: P<0.001; parental exposure×larval exposure: P=0.815), or two-tailed t-test (C: within control: P=0.941) or Mann–Whitney rank sum test (B: within control: P=0.394, within combined exposure: P=0.937; C: within combined exposure: P=0.002) across exposure at 5 dpf, followed by two-tailed t-tests (B: offspring control: P=0.987, C: offspring Control: P<0.001, offspring combined exposure: P=0.015) or Mann–Whitney rank sum tests (B: offspring combined exposure: P=0.394) between parental treatments. Differences within treatments across exposure at 5 dpf are indicated by different letters. Differences between treatments for a given exposure at 5 dpf are indicated by an asterisk. Data are means±s.e.m. in A, and medians and interquartile ranges in B and C (all n=6, except HSP70 for combined exposure larvae from control parents, n=5).

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 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.