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Figure S1. Adult gill NEC density in wild-type and Hif-1�� knockout fish. Welch Two Sample t-test (t=-1.4, p=0.19, n=7 for wild-type and n=6 for Hif-1��-/-).

Hypoxia-inducible factor 1-�� (Hif-1��), an important transcription factor regulating cellular responses to reductions in O2, previously was shown to improve hypoxia tolerance in zebrafish (Danio rerio). Here, we examined the contribution of Hif-1�� to hypoxic survival, focusing on the benefit of aquatic surface respiration (ASR). Wild-type and Hif-1�� knockout lines of adult zebrafish were exposed to two levels (moderate or severe) of intermittent hypoxia. Survival was significantly compromised in Hif-1�� knockout zebrafish prevented from accessing the surface during severe (16 mmHg) but not moderate (23 mmHg) hypoxia. When allowed access to the surface in severe hypoxia, survival times did not differ between wild-type and Hif-1�� knockouts. Performing ASR mitigated the negative effects of the loss of Hif-1�� with the knockouts initiating ASR at a higher PO2 threshold and performing ASR for longer than wild-types. The loss of Hif-1�� had little impact on survival in fish between 1 and 5 days post-fertilization, but as the larvae aged, their reliance on Hif-1�� increased. Similar to adult fish, ASR compensated for the loss of Hif-1�� on survival. Together, these results demonstrate that age, hypoxia severity and, in particular, the ability to perform ASR significantly modulate the impact of Hif-1�� on survival in hypoxic zebrafish.

Figure S2. Percent survival of wild-type and Hif-1��-/- larval zebrafish in normoxia and intermittent hypoxia with and without access to the surface. Larval zebrafish were exposed to normoxia (A, C, F), or intermittent hypoxia (16 mmHg for 4 h per day) without access to the surface (D, G), or with access to the surface (B,E, H). Individuals of each genotype were tested from 1 to 5 days post fertilization (dpf) (A,B), 5 to 9 dpf (C-E), and 12 to 16 dpf (F-H). Data points to the left of the dashed line represent survival at the onset of experiment and the first data point to the right of the dashed line represents survival following first hypoxic bout or equivalent time for fish that remained in normoxia. Larval zebrafish from 1 to 5 dpf were not tested in intermittent hypoxia without access to the surface. Statistical analysis are presented in Figure 3. Data are presented as means��s.e.m.

Figure S1. O2 consumption (ṀO2) across a range of water PO2 in wild-type (A), Hif1aa-/- (B), Hif1ab-/- (C) and Hif1aa-/-ab-/- (D) 7 day post fertilization (dpf) larvae reared in normoxia, 12 kPa hypoxia from 4 to 7 dpf and 4 kPa hypoxia from 6 to 7 dpf.;Figure S2. O2 consumption (ṀO2) across a range of water PO2 in wild-type (A), Hif1aa-/- (B), Hif1ab-/- (C) and Hif1aa-/-ab-/- (D) adult zebrafish. ;Table S1. Comparison of critical O2 tension (Pcrit) calculated using broken stick regression (BSR) and as a linear function of ṀO2 measured at low PO2 (LLO) in wild- type, Hif1aa-/-, Hif1ab-/- and Hif1aa-/-ab-/- in 7 days post fertilization larval zebrafish reared under three different O2 conditions: normoxia, 12 kPa and 4 kPa.;Table S2. Comparison of critical O2 tension (Pcrit) calculated using broken stick regression (BSR) and as a linear function of ṀO2 measured at low PO2 (LLO) in wild-type, Hif1aa-/-, Hif1ab-/- and Hif1aa-/-ab-/- in adult zebrafish.