Acute embryonic anoxia exposure favours the development of a dominant and aggressive phenotype in adult zebrafish

Abstract

Eutrophication and climate change are increasing the incidence of severe hypoxia in fish nursery habitats, yet the programming effects of hypoxia on stress responsiveness in later life are poorly understood. In this study, to investigate whether early hypoxia alters the developmental trajectory of the stress response, zebrafish embryos were exposed to 4 h of anoxia at 36 h post-fertilization and reared to adults when the responses to secondary stressors were assessed. While embryonic anoxia did not affect basal cortisol levels or the cortisol response to hypoxia in later life, it had a marked effect on the responses to a social stressor. In dyadic social interactions, adults derived from embryonic anoxia initiated more chases, bit more often, entered fewer freezes and had lower cortisol levels. Adults derived from embryonic anoxia also performed more bites towards their mirror image, had lower gonadal aromatase gene expression and had higher testosterone levels. We conclude that acute embryonic anoxia has long-lasting consequences for the hormonal and behavioural responses to social interactions in zebrafish. Specifically, we demonstrate that acute embryonic anoxia favours the development of a dominant and aggressive phenotype, and that a disruption in sex steroid production may contribute to the programming effects of environmental hypoxia.

Keywords: aggression; developmental programming; dyadic interactions; hypoxia; stress response; zebrafish.

Figure 1.

Effects of exposing 36 hpf embryos to either 4 h of normoxia or anoxia on subsequent basal (normoxia) and hypoxia-induced whole-body cortisol levels in adult zebrafish. In the adult hypoxia treatment, fish were exposed to 5% DO for 10 min. Values are mean + s.e.m. (n = 13). Significant differences are indicated by dissimilar letters (two-way ANOVA and Tukey's post hoc test, p < 0.05).

Figure 2.

Effects of exposing 36 hpf embryos to 4 h of either normoxia or anoxia on subsequent behavioural responses to dyadic social interactions in adult zebrafish. Pairs of adult zebrafish consisting of one fish each from the normoxia and anoxia embryonic treatments were allowed to interact for 1 h. The dyadic social interactions were scored for (a) chases initiated, (b) bites given and (c) freezes entered. Values are mean + s.e.m. (n = 10). A significant difference is indicated by an asterisk (Welch two-sample t-test, p < 0.05).

Figure 3.

Effects of exposing 36 hpf embryos to 4 h of either normoxia or anoxia on subsequent whole-body cortisol response to dyadic social interactions in adult zebrafish. (a) Pairs of adult zebrafish consisting of one fish each from the normoxia and anoxia embryonic treatments were allowed to interact for 1 h (n = 10). A significant difference is indicated by an asterisk (Welch two-sample t-test, p < 0.05). (b) Based on a behavioural assessment of the dyadic social interactions, each fish within a pair was assigned a dominant or subordinate status. A separate group of control adult fish from the normoxia embryonic treatment (n = 7) was used to quantify whole-body cortisol levels in the absence of dyadic social interactions. Values are mean + s.e.m. Significant differences are indicated by dissimilar letters (one-way ANOVA and Tukey's post hoc test, p < 0.05).

Figure 4.

Effects of exposing 36 hpf embryos to 4 h of either normoxia or anoxia on subsequent number of bites at mirror in adult zebrafish during a 25 min mirror aggression test. Values are mean + s.e.m. (n = 11–12). A significant difference is indicated by an asterisk (Welch two-sample t-test, p < 0.05).

Figure 5.

Effects of exposing 36 hpf embryos to 4 h of either normoxia or anoxia on subsequent mRNA levels of gonadal aromatase (cyp19a) in (a) female and (b) male adult zebrafish. The mRNA levels are normalized with ef1a and presented relative to the normoxia treatment. Values are mean + s.e.m. (n = 12). A significant difference is indicated by an asterisk (Welch two-sample t-test, p < 0.05).

Figure 6.

Effects of exposing 36 hpf embryos to 4 h of either normoxia or anoxia on subsequent whole-body testosterone levels of (a) control adult zebrafish (baseline; n = 12) or adult zebrafish allowed to interact for 1 h in a dyadic social interaction contest (n = 10). Significant differences between the baseline and the dyadic interactions of adult fish are indicated by dissimilar letters, and a significant difference between embryonic treatments within a group of adult fish is indicated by an asterisk (two-way ANOVA and Tukey's post hoc test, p < 0.05). Baseline values are also shown for (b) female (n = 6) and (c) male adult fish (n = 6). A significant difference between embryonic treatments within a gender is indicated by an asterisk (Welch two-sample t-test, p < 0.05). Values are mean + s.e.m.