|Abstract||Global declines in honeybees have been linked to widespread use of pesticides. Sublethal doses of the neonicotinoid pesticide imidacloprid have been shown to cause physiological and behavioral changes that negatively impact hive health. This study examined the effects of acute, sublethal doses of imidacloprid on the honeybee gene expression in the brain.
Experiment 1 identified an imidacloprid dosage that yielded a cellular stress response in honeybees. Honeybee foragers were harnessed, fed to satiation, and randomly assigned to control (1.5 M sucrose) or treatment groups receiving imidacloprid at doses of 1/5th, 1/10th, 1/20th, 1/50th, 1/100th, and 1/500th of the LD50 (18.0 ng/bee). After four hours, impaired motor responses and elevated levels of Heat Shock Protein 70 and Superoxide Dismutase, markers of cellular and oxidative stress, respectively, were observed at sublethal imidacloprid doses. A conservative dose of 0.9 ng/bee (1/20th of the LD50) was selected to treat bees for RNA transcriptome analysis in Experiment 2.
In Experiment 2, bees were randomly assigned to four acute-exposure groups: Control-0h, Control-4h, Treatment-0h, and Treatment-4h. Control bees were fed 1.5 M sucrose while treatment groups received 0.9 ng/bee of imidacloprid. RNA isolated from bee brain tissue was sent to the University of Illinois at Urbana-Champaign for RNA sequencing. Of the 10,597 genes recovered from the reference genome, 4,205 genes were differentially expressed. Collectively, the Differential Expression Analysis, Multidimensional Scaling Plot, and heat map agree that the Control-4h group had the greatest changes in gene expression, counter to our prediction that the greatest alteration would be in imidacloprid-treated bees. Corroborating evidence supported the post-hoc hypothesis that the Control-4h and the Treatment-4h samples were switched and mislabeled prior to shipping. Comparisons between Control-4h and Treatment 4-h groups remain valid. If samples were switched, only the direction of differential gene expression (i.e., up-regulation versus down-regulation) would be affected by imidacloprid. Gene set enrichment analysis indicated that the key pathways affected were: oxidative phosphorylation, longevity regulating pathway, apoptosis, peroxisome, FOXO signaling, drug metabolism- cytochrome P450, metabolism of xenobiotics by cytochrome P450, circadian rhythm, and glutathione metabolism. These gene networks relate to key biological functions of honeybees that have the potential to affect colony viability. Future research will focus on hypothesis-driven gene expression studies that relate specific molecular changes to biological functions and organism-level performance, an integrative approach that is essential to understanding the declines of these essential pollinators. |