Assessing the Pcrit in relation to temperature and the expression of hypoxia associated genes in the mayfly, Neocloeon triangulifer.

Hypoxia is a growing concern in aquatic ecosystems. Historically, scientists have used the Pcrit (the dissolved oxygen level below which an animal can no longer oxyregulate) to infer hypoxia tolerance across species. Here, we tested the hypothesis that the Pcrit is positively correlated with temperature in the mayfly, Neocloeon triangulifer. Cross-temperature comparisons showed a modest (r = 0.47), but significant (p < 0.0001) association between temperature and Pcrit despite relatively large interindividual variability (Coefficient of Variance (CV) = 39.9% at 18 °C). We used the expression of hypoxia-responsive genes EGL-9 (an oxygen sensing gene and modulator of HIF-1a activity) and LDH (a hypoxia indicator) to test whether oxygen partial pressure near the Pcrit stimulates expression of hypoxia-responsive genes. Neither gene was upregulated at oxygen levels above the estimated Pcrit, however, at or below the Pcrit estimates, expression of both genes was stimulated (~20- and ~3-fold change for EGL-9 and LDH, respectively). Finally, we evaluated the influence of hypoxic exposure time and pretreatment conditions on the mRNA expression levels of hypoxia-responsive genes. When larvae were exposed to a gradual reduction of DO, hypoxic gene expression was more robust than during instantaneous exposure to hypoxia. Our data provide modest support for traditional interpretation of the Pcrit as a physiologically meaningful shift from aerobic to anaerobic metabolism in N. triangulifer. However, we also discuss limitations of the Pcrit as a proxy measure of hypoxia tolerance at the species level.