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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.

The current study investigated the role of hydrogen sulphide (H2S) in oxygen sensing, intracellular signalling and promotion of ventilatory responses to hypoxia in adult and larval zebrafish (Danio rerio). Both larval and adult zebrafish exhibited a dose-dependent increase in ventilation to sodium sulphide (Na2S), an H2S donor. In vertebrates, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are enzymes that catalyse the endogenous production of H2S. In adult zebrafish, inhibition of both CBS and CSE with aminooxyacetate (AOA) and propargyl glycine (PPG) blunted or abolished the hypoxic hyperventilation, and the addition of Na2S to the water partially rescued the effects of inhibiting endogenous H2S production. In zebrafish larvae (4 days post-fertilization), gene knockdown of either CBS or CSE using morpholinos attenuated the hypoxic ventilatory response. Furthermore, the intracellular calcium concentration of isolated neuroepithelial cells (NECs), which are putative oxygen chemoreceptors, increased significantly when these cells were exposed to 50 μm Na2S, supporting a role for H2S in Ca(2+)-evoked neurotransmitter release in these cells. Finally, immunohistochemical labelling showed that NECs dissociated from adult gill contained CBS and CSE, whereas cutaneous NECs in larval zebrafish expressed only CSE. Taken together, these data show that H2S can be produced in the putative oxygen-sensing cells of zebrafish, the NECs, in which it appears to play a pivotal role in promoting the hypoxic ventilatory response.