Sublethal effects of imidacloprid on honeybee stress response and circadian rhythm

Honeybees are important pollinators, essential to worldwide food supply. These insects are experiencing global declines, an effect that may be linked to the widespread agricultural use of neonicotinoid pesticides. Sublethal doses of the common neonicotinoid pesticide imidacloprid result in physiological and behavioral changes that negatively impact hive health. This research investigates the effect of imidacloprid on stress and circadian rhythm cellular pathways. We studied cellular stress responses ( e.g., superoxide dismutase, heat shock protein 70) and qPCR of genes involved in stress and circadian rhythm pathways (e.g., period, heat shock protein 70) reported to be affected in an previous RNA-seq study. Additional markers of stress were also analyzed including heat shock factor 1, and nuclear factor erythroid 2 related factor 2. In late summer of 2021, two different subspecies of honeybee, Apis mellifera (mixed Russian x Italian honeybee genetics) and Apis mellifera carnica (Carniolan) were collected locally and randomly assigned control or treatment with a sublethal dose of imidacloprid, a harmful neonicotinoid pesticide. After being harnessed for 22-24 h, control bees were fed 10l of
1.5 M sucrose and treatment bees were fed 0.9 ng imidacloprid/bee in a 10l volume. At either zero or four hours post treatment, head capsules were excised and homogenized in buffer solution for analysis of superoxide dismutase (SOD) enzyme activity and heat shock protein 70 (HSP70) levels. Three-way ANOVA analysis revealed no significant difference between control and treatment bees for SOD activity (F=0.599, df=7,110, P=0.756). When examining HSP70, the two subspecies did differ in the heat shock response (F=3.580, df=7,110, P= 0.0017). Gene expression analysis was also conducted using qPCR methods. Genes encoding per, hsp70, hsf1, and sod increased in expression for both subspecies. Nrf2 was found to be upregulated in A. mellifera and did not change in A. m. carnica. The only gene that was significantly upregulated was hsp70 (P=0.021). This suggests a sublethal dose of imidacloprid can have biologically significant effects on honeybee circadian rhythmicity and stress response.