Off-target stoichiometric binding identified from toxicogenomics explains why some species are more sensitive than others to a widely used neonicotinoid

Neonicotinoids are currently licensed for use in 120 countries, making accurate nontarget species sensitivity predictions critical. Unfortunately, such predictions are fraught with uncertainty, as sensitivity is extrapolated from only a few test species and neonicotinoid sensitivities can differ greatly between closely related taxa. Combining classical toxicology with de novo toxicogenomics could greatly improve sensitivity predictions and identify unexpectedly susceptible species. We show that there is a >30-fold differential species sensitivity (DSS) for the neonicotinoid imidacloprid between five earthworm species, a critical nontarget taxon. This variation could not be explained by differential toxicokinetics. Furthermore, comparing key motif expression in subunit genes of the classical nicotinic acetylcholine receptor (nAChR) target predicts only minor differences in the ligand binding domains (LBDs). In contrast, predicted dissimilarities in LBDs do occur in the highly expressed but nonclassical targets, acetylcholine binding proteins (AChBPs). Critically, the predicted AChBP divergence is capable of explaining DSS. We propose that high expression levels of putative nonsynaptic AChBPs with high imidacloprid affinities reduce imidacloprid binding to critical nAChRs involved in vital synaptic neurotransmission. This study provides a clear example of how pragmatic interrogation of key motif expression in complex multisubunit receptors can predict observed DSS, thereby informing sensitivity predictions for essential nontarget species.