Integrated Assessment of Pollen Substitute Diets in Honey Bees Based on Transcriptional Responses, Immune-Derived Predicted Biological Age, and Survival

Heat events and nutritional stress threaten honey bee health, but how pollen substitute formulation shapes responses to acute heat remains unclear. Newly emerged Apis mellifera workers received three pollen substitute diets or two beebread-based reference diets for five days before exposure to 45 °C for 4 h. Expression of 12 genes related to nutrition, endocrine regulation, immunity, oxidative defense, and cellular stress was examined using mixed-effects and multivariate analyses. Immune-derived predicted biological age was estimated before and after heat exposure. Apparent consumption and survival were evaluated in an independent 18-day feeding assay without heat. Significant diet × heat interactions occurred for 10 genes, whereas tor and dome showed no significant interactions. At baseline, Diet2(M) exhibited a transcriptional profile similar to the control and a lower predicted biological age than Diet1(S) and Diet3(O). Diet2(M) also supported higher survival than Diet1(S), despite similar apparent consumption among substitute diets. These findings indicate that pollen substitute composition, rather than apparent consumption alone, can shape physiology and survival. Diet2(M) is therefore a priority candidate for colony-level evaluation, although improved heat tolerance was not demonstrated. These findings may guide the development of supplemental feeds supporting honey bee health under climate-related pollen scarcity and heat risk.