Water homeostasis and extracellular fluid dynamics reveal imidacloprid disruption and rutin mitigation in honey bees

Pollinator decline is partly driven by exposure to agrochemicals such as imidacloprid, a neonicotinoid insecticide that impairs feeding and water balance in bees. This study examines how imidacloprid and the dietary flavonoid rutin, individually and combined, affect body mass regulation and extracellular fluid in Apis mellifera. Africanized honey bees were exposed to 0.3, 0.6, or 1.0 ppm of each substance. We conducted four experiments to evaluate short- and long-term changes in food intake, wet and dry mass, hydration, and fluid evaporation rates. Imidacloprid consistently reduced food consumption and body mass gain, while rutin promoted weight gain, increased water retention, and partially reversed imidacloprid's effects. The combination treatment produced intermediate results. Notably, extracellular fluid from imidacloprid-treated bees showed significantly slower evaporation, suggesting altered physicochemical properties. This represents the first application of extracellular fluid evaporation as a physiological biomarker of sublethal stress in honey bees, expanding current approaches beyond conventional endpoints such as survival or behavior. These physiological changes persisted beyond the exposure period in some cases. Our results highlight the potential of mass dynamics and fluid behavior as sensitive markers of sublethal stress. Rutin appears to enhance physiological resilience, supporting its role as a candidate dietary mitigator. This work contributes to the development of new physiological endpoints for assessing the impact of agrochemicals on pollinators and offers insight into the protective role of plant-derived compounds.