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

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Resistance to insecticides through enhanced metabolism is a worldwide problem. The Cyp6g1 gene of the vinegar fly, Drosophila melanogaster, is a paradigm for the study of metabolic resistance. Constitutive overexpression of this gene confers resistance to several classes of insecticides, including the neonicotinoid imidacloprid (IMI). The metabolism of IMI in this species has been previously shown to yield oxidative and nitro-reduced metabolites. While levels of the oxidative metabolites are correlated with CYP6G1 expression, nitro-reduced metabolites are not, raising the question of how these metabolites are produced. Some IMI metabolites are known to be toxic, making their fate within the insect a second question of interest. These questions have been addressed by coupling the genetic tools of gene overexpression and CRISPR gene knock-out with the mass spectrometric technique, the Twin-Ion Method (TIM). Analysing axenic larvae indicated that microbes living within D. melanogaster are largely responsible for the production of the nitro-reduced metabolites. Knock-out of Cyp6g1 revealed functional redundancy, with some metabolites produced by CYP6G1 still detected. IMI metabolism was shown to produce toxic products that are not further metabolized but readily excreted, even when produced in the Central Nervous System (CNS), highlighting the significance of transport and excretion in metabolic resistance.

One of the major insect metabolites of the widely used neonicotinoid insecticide imidacloprid, 1 (1-[(6-chloro-3-pyridinyl)methyl]-N-nitro-1H-imidazol-2-amine), is the olefin 2. To better understand how the structure of olefin 2 relates to the gas-phase fragmentation of its protonated form, 2H(+), X-ray crystallography, tandem mass spectrometry experiments and DFT calculations were carried out. Olefin 2 was found to be in a tautomeric form where the proton is on the N(1) position of the imidazole ring and forms a hydrogen bond to one of the oxygen atoms of the coplanar nitroamine group. Under conditions of low-energy collision-induced dissociation (CID) in a linear ion trap, 2H(+), formed via electrospray ionization (ESI), fragments via a major loss of water, together with minor competing losses of HNO2 and NO2•.This contrasts with 1H+, which mainly undergoes bond homolysis via NO2• loss. Thus, installation of the double bond in 2 plays a key role in facilitating the loss of water. DFT calculations, carried out using the B3LYP/6-311G++(d,p) level of theory, revealed that loss of water was energetically more favourable compared to HNO2 and NO2• loss. Three multistep, energetically accessible mechanisms were identified for loss of water from 2H(+), and these have the following barriers: (I) direct proton transfer from N(5) of the pyridine to O(1) on the NO2 group (119 kJ mol(-1)); (II) rotation of the N(2)-N(4) bond (117 kJ mol(-1)); (III) 1,3-intramolecular proton transfer between the two oxygen atoms of the NO2 group (145 kJ mol(-1)). Given that the lowest barrier for the losses of HNO2 and NO2• is 156 kJ mol(-1), it is likely that all three water loss mechanisms occur concurrently.

Abstract: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.