Genetics of tolerance in honey bees to the neonicotinoid clothianidin.

The effects of neonicotinoid insecticides (NNIs) on honey bee health is intensely debated, with numerous studies showing negative effects of exposure, while others report no such effects. Understanding the cause of these differences is critical for developing evidence-based policy on the use of NNIs. We carried out experiments to study the genetic and molecular basis of NNI tolerance in honey bees, which may underlie the discrepancies observed in the literature. We discovered that worker survival post-exposure to an acute oral dose of clothianidin is heritable (H2=37.8%). Tolerance to clothianidin was not associated with differences in expression of detoxification enzymes in our experiments. Instead, mutations in the primary neonicotinoid detoxification genes CYP9Q1 and CYP9Q3 were strongly associated with worker survival post clothianidin exposure. In some instances, the strong association between CYP9Q haplotypes and worker survival was associated with the protein’s predicted binding affinity for clothianidin. Our findings have implications regarding future toxicological studies utilizing honey bees as a model pollinator. Eight day old bees were exposed to 4.27ppb of clothianidin individually through a sugar solution. 24 Hours post exposure, the bees were frozen on dry ice. The patriline of each bee was determined using DNA microsatalites. 'suseptible' and tolorant' patrilines were determined based on a previous experiment described in the manuscript. We performed dissections of the brain, Malpighian tubules, and ventriculus and kept them at -80oC until RNA could be extracted. RNA extractions were completed using the miRNeasy Mini kit (Qiagen). The ventriculus, brains, and Malpighian tubules of 5 individual bees from each patriline were pooled separately based on their organ (e,g., all the brains of a patriline were pooled together). We had a total of 3 tolerant patrilines, and 3 susceptible patrilines and 18 pools in total. To ensure equal representation for each bee, we added 300 ng of RNA to each pool (1500 ng/5 bees). Library preparation and 100 bp pair-end sequencing using the Illumina NovaSeq 6000 S4 were carried out by Genome Quebec’s sequencing facility (Montreal, QC).