Clodronate liposomes untangle the role of hemocytes in Apis mellifera response to temperature variation and microbial infection.

Introduction: The cellular immune response, mediated by hemocytes, is a fundamental component of honey bee (Apis mellifera) health. However, the specific contribution of hemocyte subtypes to resilience against combined stressors remains poorly characterized. This study investigated the effect of temperature and bacterial challenge on hemocyte abundance. We employed clodronate liposomes (CLD) for the first time in honey bees as a novel tool for the selective depletion of hemocytes to investigate this gap. Methods: Five-day-old (nurses) and fifteen-day-old (foragers) honey bees were treated with CLD, control liposomes, PBS, or left untreated, then exposed at either 32°C or 22°C and challenged with the gram-negative bacterium, Escherichia coli, or the gram-positive bacterium, Staphylococcus aureus. Survival, hemolymph volume, total hemocyte counts, and differential hemocyte counts were monitored over seven days from the start of exposure. Results: The CLD application demonstrated significant reductions in granulocyte and prohemocyte populations, indicating the highest vulnerability. A temperature drop to 22°C buffered the negative impact on survival of CLD-induced immunosuppression. While bacterial challenges universally reduced hemocyte counts, we found an age-dependent difference where nurses maintained significantly higher baseline total hemocyte counts than foragers. Furthermore, temperature did not affect overall total hemocyte counts in 5-day-old nurse bees, but in 15-day-old foragers, it significantly modulated the hemocyte response to bacterial infection. Conclusion: Our findings show that hemocyte function is non-uniform, with specific subtypes being essential for overall resilience. The results highlight a previously underappreciated role for temperature as a key modulator of immune capacity, particularly in immunocompromised bees. The age-related differences in hemocyte abundance suggest a life-history trade-off that may prompt the increased vulnerability of honey bees as they age. This work establishes CLD as a powerful tool for insect immunology and sets a precedent for using precise immune manipulation to study host-pathogen-environment interactions.