Combined effect of temperature and ammonia on molecular response and survival of the freshwater crustacean Gammarus pulex

Freshwater ecosystems are experiencing mounting pressures from agriculture, urbanization, and climate change, which could drastically impair aquatic biodiversity. As nutrient inputs increase and temperatures rise, ammonia (NH₃) concentration is likely to be associated with stressful temperatures. To investigate the interaction between NH₃ and temperature on aquatic invertebrate survival, we performed a factorial experiment on the survival and molecular response of Gammarus pulex, with temperature (10, 15, 20, and 25°C) and NH₃ (0, 0.5, 1, 2, 3, and 4mg NH₃/L) treatments. We observed an unexpected antagonistic interaction between temperature and NH₃ concentration, meaning survival in the 4mg NH₃/L treatment was higher at 25°C than at the control temperature of 10°C. A toxicokinetic-toxicodynamic (TK-TD) model was built to describe this antagonistic interaction. While the No Effect Concentration showed no significant variation across temperatures, the 50% lethal concentration at the end of the experiment increased from 2.7 (2.1-3.6) at 10°C to 5.5 (3.5- 23.4) mg NH₃/L at 25°C. Based on qPCR data, we associated these survival patterns to variations in the expression of the hsp70 gene, a generic biomarker of stress. However, though there was a 14-fold increase in hsp70 mRNA expression for gammarids exposed to 25°C compared to controls, NH₃ concentration had no effect on hsp70 mRNA synthesis across temperatures. Our results demonstrate that the effects of combined environmental stressors, like temperature and NH₃, may strongly differ from simple additive effects, and that stress response to temperature can actually increase resilience to nutrient pollution in some circumstances.