Declining insect population sizes are provoking grave concern around the world as insects play essential roles in food production and ecosystems. Environmental contamination by intense insecticide usage is consistently proposed as a significant contributor, among other threats. Many studies have demonstrated impacts of low doses of insecticides on insect behavior, but have not elucidated links to insecticidal activity at the molecular and cellular levels. Here, the histological, physiological, and behavioral impacts of imidacloprid are investigated in Drosophila melanogaster, an experimental organism exposed to insecticides in the field. We show that oxidative stress is a key factor in the mode of action of this insecticide at low doses. Imidacloprid produces an enduring flux of Ca2+ into neurons and a rapid increase in levels of reactive oxygen species (ROS) in the larval brain. It affects mitochondrial function, energy levels, the lipid environment, and transcriptomic profiles. Use of RNAi to induce ROS production in the brain recapitulates insecticide-induced phenotypes in the metabolic tissues, indicating that a signal from neurons is responsible. Chronic low level exposures in adults lead to mitochondrial dysfunction, severe damage to glial cells, and impaired vision. The potent antioxidant, N-acetylcysteine amide (NACA), reduces the severity of a number of the imidacloprid-induced phenotypes, indicating a causal role for oxidative stress. Given that other insecticides are known to generate oxidative stress, this research has wider implications. The systemic impairment of several key biological functions, including vision, reported here would reduce the resilience of insects facing other environmental challenges.

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Insecticide resistance is an economically important example of evolution in response to intense selection pressure. Here, the genetics of resistance to the neonicotinoid insecticide imidacloprid is explored using the Drosophila Genetic Reference Panel, a collection of inbred Drosophila melanogaster genotypes derived from a single population in North Carolina. Imidacloprid resistance varied substantially among genotypes, and more resistant genotypes tended to show increased capacity to metabolize and excrete imidacloprid. Variation in resistance level was then associated with genomic and transcriptomic variation, implicating several candidate genes involved in central nervous system function and the cytochrome P450s Cyp6g1 and Cyp6g2. CRISPR-Cas9 mediated removal of Cyp6g1 suggested that it contributed to imidacloprid resistance only in backgrounds where it was already highly expressed. Cyp6g2, previously implicated in juvenile hormone synthesis via expression in the ring gland, was shown to be expressed in metabolically relevant tissues of resistant genotypes. Cyp6g2 overexpression was shown to both metabolize imidacloprid and confer resistance. These data collectively suggest that imidacloprid resistance is influenced by a variety of previously known and unknown genetic factors.The introduction of synthetic insecticides is often followed by the appearance of resistance phenotypes in field populations, leading to significant reductions in agricultural production 1 . There has been much debate about whether the evolution of resistance is caused by variation in a single gene (monogenic) or by the additive effects of many (polygenic) 2, 3 . Substantially more work has been dedicated to characterizing the monogenic variants, but such alleles arise in a genetic background where there is polygenic variation for tolerance to the insecticide 2 . Much still remains unclear about the relative contribution of different alleles to insecticide resistance, but D. melanogaster is uniquely placed to answer such questions, owing to the extensive genetic toolkit that has been developed in this model insect.Imidacloprid is amongst the most widely used insecticides. It is derived from nicotine and is a member of the neonicotinoid chemical class. Neonicotinoids target nicotinic acetylcholine receptors (nAChRs) that have vital roles in neurotransmission and behaviour in insects 4, 5 . However, imidacloprid resistance via mutations in targets is not the most common resistance mechanisms, possibly due to associated fitness costs 6 . The overexpression of cytochrome P450 enzymes (P450s) more frequently underpins imidacloprid resistance 7 . Some members of the P450 superfamily can function as drug metabolizing enzymes (DMEs) with xenobiotic substrates, while others have vital roles in development using endogenous substrates 8 . P450s which are capable of metabolizing imidacloprid and conferring resistance have been identified in several species [9][10][11] ; the Cyp6g1 gene of D. melanogaster

Large-scale insecticide application is a primary weapon in the control of insect pests in agriculture. However, a growing body of evidence indicates that it is contributing to the global decline in population sizes of many beneficial insect species. Spinosad emerged as an organic alternative to synthetic insecticides and is considered less harmful to beneficial insects, yet its mode of action remains unclear. Using Drosophila, we show that low doses of spinosad antagonize its neuronal target, the nicotinic acetylcholine receptor subunit alpha 6 (nAChRα6), reducing the cholinergic response. We show that the nAChRα6 receptors are transported to lysosomes that become enlarged and increase in number upon low doses of spinosad treatment. Lysosomal dysfunction is associated with mitochondrial stress and elevated levels of reactive oxygen species (ROS) in the central nervous system where nAChRα6 is broadly expressed. ROS disturb lipid storage in metabolic tissues in an nAChRα6-dependent manner. Spinosad toxicity is ameliorated with the antioxidant N-acetylcysteine amide. Chronic exposure of adult virgin females to low doses of spinosad leads to mitochondrial defects, severe neurodegeneration, and blindness. These deleterious effects of low-dose exposures warrant rigorous investigation of its impacts on beneficial insects.

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