Significance: Intense insecticide usage is suggested to be a significant contributor to the observed decline of insect populations around the world. Beneficial insects play essential roles in food production and ecosystem health. It is therefore vital to understand the mechanisms by which low doses of insecticide impact insect biology in order to understand and assess the threat posed. We investigated the impacts of the neonicotinoid insecticide imidacloprid on Drosophila . The binding of the insecticide to receptors in the brain triggers oxidative stress, reduces energy levels, and induces neurodegeneration as well as vision loss. As the receptors targeted by imidacloprid are conserved among insects, and other insecticides have also been shown to cause oxidative stress, these findings have wider significance.

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.

BACKGROUND: The insecticides spinosad and imidacloprid are neurotoxins with distinct modes of action. Both target nicotinic acetylcholine receptors (nAChRs), albeit different subunits. Spinosad is an allosteric modulator, that upon binding initiates endocytosis of its target, nAChRα6. Imidacloprid binding triggers excessive neuronal ion influx. Despite these differences, low‐dose effects converge downstream in the precipitation of oxidative stress and neurodegeneration.RESULTS: Using RNA‐sequencing, we compared the transcriptional signatures of spinosad and imidacloprid, at low‐dose exposures. Both insecticides cause up‐regulation of glutathione S‐transferase and cytochrome P450 genes in the brain and down‐regulation in the fat body, whereas reduced expression of immune‐related genes is observed in both tissues. Spinosad shows unique impacts on genes involved in lysosomal function, protein folding, and reproduction. Co‐expression analyses revealed little to no correlation between genes affected by spinosad and nAChRα6 expressing neurons, but a positive correlation with glial cell markers. We also detected and experimentally confirmed nAChRα6 expression in fat body cells and male germline cells. This led us to uncover lysosomal dysfunction in the fat body following spinosad exposure, and a fitness cost in spinosad‐resistant (nAChRα6 null) males – oxidative stress in testes, and reduced fertility.CONCLUSION: Spinosad and imidacloprid share transcriptional perturbations in immunity‐, energy homeostasis‐, and oxidative stress‐related genes. Low doses of other neurotoxic insecticides should be investigated for similar impacts. While target‐site spinosad resistance mutation has evolved in the field, this may have a fitness cost. Our findings demonstrate the power of tissue‐specific transcriptomics approach and the use of single‐cell transcriptome data. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.