Fishes are frequently exposed to hypoxic stress, yet their tolerance to hypoxia varies significantly among species. The association between this variation and alterations in the hypoxia-inducible factor (HIF) pathway remains unclear. We discovered that otomorphs generally retain two Hif-1α paralogs (Hif-1αa and Hif-1αb), resulting from the teleost-specific genome duplication (TGD), whereas most euteleosts possess only a single Hif-1αa copy. In otomorphs, key mutations disrupt one conserved Leu-X-X-Leu-Ala-Pro (LXXLAP) motif in the oxygen-dependent degradation (ODD) domain of the Hif-1αa proteins. Molecular dynamics simulations revealed that these mutations impede the recognition of the critical proline residue by prolyl hydroxylase domain protein 2 (PHD2), suggesting enhanced normoxic stability of Hif-1αa. We also investigated the expression profiles of hif-1α and downstream genes in four fish species (two otomorphs and two euteleosts). In otomorphs, the hif-1αa genes were highly expressed specifically in the heart; concomitantly, two critical downstream genes, ldha and mct4, exhibited relatively high expression levels in vital tissues such as the heart, brain, and muscle. This coordinated expression pattern promotes a heightened glycolytic capacity and facilitates lactate shuttling in these tissues, thereby ensuring energy supply during hypoxic stress. Our integrated computational analyses indicate that otomorphs achieve enhanced hypoxia tolerance through the subfunctionalization of Hif-1α paralogs.

To explore the molecular basis of hypoxia tolerance variation within euteleosts, we compared the hypoxia-inducible factor (HIF) pathways of the highly tolerant Nile tilapia (Oreochromis niloticus) and the hypoxia-sensitive rainbow trout (Oncorhynchus mykiss). Evolutionary analysis revealed that Nile tilapia possesses single copies of Hif-1α and prolyl hydroxylase domain protein 2 (Phd2), whereas rainbow trout retains two and three copies, respectively. The Leu-X-X-Leu-Ala-Pro (LXXLAP) motifs in the oxygen-dependent degradation (ODD) domain of Hif-1α and the interacting loop region of Phd2 are highly conserved, indicating a conserved core mechanism for regulating Hif-1α stability. However, differences in charged residue composition flanking the Phd2 loop (e.g., fewer positively charged residues in Nile tilapia) were identified. Molecular dynamics simulations revealed that the complex formed by Nile tilapia Phd2 and the Hif-1α LXXLAP motif was unstable across physiological temperatures, suggesting potential impairment of the catalytic geometry compatible with hydroxylation and elevated normoxic Hif-1α stability. In contrast, the corresponding complexes in rainbow trout were more stable, particularly at low temperatures. Expression profiling revealed that Nile tilapia tissues, including the heart, maintain higher basal expression of glycolytic genes, may help support energy production during hypoxia. Our findings indicate that a weakened protein interaction and high constitutive expression is predicted to enhance HIF pathway responsiveness, potentially priming vital tissues for glycolytic energy production and may contribute to this species’ hypoxia tolerance.