The increasing frequency and intensity of summer heat waves is pushing freshwater zooplankton towards their upper thermal tolerance limits. At the same time, higher temperatures and prolonged water column stratification can favor the dominance of cyanobacteria in phytoplankton. Even when not toxic or grazing resistant, these prokaryotes lack phytosterols as essential precursors for cholesterol, the main sterol in animal tissues. Cholesterol plays a crucial role in the physiological adaptation of ectotherms to high temperature. Therefore, the shift to cyanobacteria-dominated systems may increase the vulnerability of zooplankton to heatwaves by intensifying cholesterol limitation. Here, we used death time curves that take into consideration the intensity and duration of a thermal challenge and a dynamic model to study the effects of cholesterol limitation on the heat tolerance of the keystone species Daphnia magna and to simulate the cumulative mortality that could occur in a fluctuating environment over several days of heatwave.  We show that increasing cholesterol limitation does not affect the slope between time-to-immobilization and temperature, but does decrease the maximal temperature that Daphnia can withstand by up to 0.74°C. This seemingly small difference is sufficient to halve the time individuals can survive heat stress.  Our simulations predicted that, when facing heatwaves over several days, the differences in survival caused by cholesterol limitation build up rapidly. Considering the anticipated intensity and duration of future (2070-2099) heatwaves, cholesterol limitation could increase mortality by up to 45% and 72% under low- and medium-greenhouse-gas-emission scenarios, respectively. These results suggest that the increasing risk of cholesterol limitation due to more frequent cyanobacterial blooms could compromise the resistance of zooplankton populations to future heatwaves. More generally, this study shows the importance of considering the nutritional context in any attempt to predict ectotherm mortality with increasing temperatures in the field.

We used death time curves to study the effects of cholesterol limitation on the heat tolerance of the keystone species Daphnia magna and to simulate the cumulative mortality that could occur in a fluctuating environment over several days of heatwave. In that aim, D. magna neonates were grown under controlled laboratory condition on a sterol-free, non-toxic cyanobacterial diet (Synnechococcus obliquus) supplemented with different amounts of cholesterol-containing liposomes to obtain four cholesterol concentrations of 0.25, 2.5, 5, and 8 µg per mg C. In all treatments, a small amount of eicosapentaenoic acid (20:5ω3) was also added via liposomes (resulting in 0.25 µg 20:5ω3 per mg C) in order to prevent severe 20:5ω3 limitation, thereby aiding the development of daphnids. The daphnids were grown at 27°C, a non-lethal yet super-optimal temperature. Heat tolerance (time to immobilization) of the daphnids from the different cholesterol treatments was tested on day seven at five different constant temperatures (34.5°, 35.5°, 36.5°, 37.5°, 38.5°C). Here, we provide the raw data, as well as the R scripts used to analyze the data.