Clodronate Liposomes Untangle the Role of Hemocytes in Apis mellifera Response to Temperature Variation and Microbial...

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.

Five-day-old (nurses) and fifteen-day-old (foragers) honey bees were treated with CLD, control liposomes, phosphate-buffered saline, injection wound (sham), 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 7 days from the start of exposure.

The CLD application demonstrated significant reductions in granulocyte and prohemocyte populations. This indicates that the granulocytes are the phagocytic cell type and prohemocytes are probably used to replenish the granulocytes. A temperature drop to 22°C buffered the negative impact of CLD-induced immunosuppression on honey bee survival. While bacterial challenge universally reduced hemocyte counts, we found an age-dependent difference, where young maintained significantly higher baseline total hemocyte counts than older bees. Furthermore, temperature did not affect overall total hemocyte counts in 5-day-old bees but did in 15-day-old bees, where it significantly modulated the hemocyte response to bacterial infection.

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 decreases in hemocyte abundance, coupled with increased survival after an immune challenge, suggests a life history trade-off where older bees rely less on cellular immunity and more on other physiological mechanisms for resilience. 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.