Investigating resistance to neonicotinoid insecticides in insect pests

Neonicotinoids represent the fastest-growing class of insecticides introduced for pest management since the commercialisation of pyrethroids. Their sales worldwide are already estimated to exceed one billion US dollars per year. These novel, safe and highly effective compounds provide a invaluable respite from problems of pest resistance to earlier-used chemicals, but are themselves very vulnerable to appearance of new resistance mechanisms. Widespread establishment of neonicotinoid resistance would severely compromise the environmental and economic sustainability of crop protection strategies in many parts of the world including the UK. The brown rice planthopper (Nilaparvata lugens) and the tobacco whitefly (Bemisia tabaci) are two of the relatively few species to have developed strong neonicotinoid resistance to date. Such species serve as ideal models for analysing the underlying mechanisms and their practical implications, and for forewarning of potential problems in a wider range of pest species. Little is still known about the mechanisms that could confer protection from neonicotinoids. However, in N. lugens we have identified a mutation in the nicotinic acetylcholine receptor (nAChR), the protein in the insect nervous system targeted by neonicotinoids, which greatly reduces binding between insecticides and the receptor and confers substantially reduced susceptibility in whole-organism bioassays. Since target-site resistance mutations for other insecticide classes have often proved to be identical across species, results from planthoppers are likely to be transferable to other major targets of neonicotinoids. This project will exploit established, international monitoring networks to obtain samples of N. lugens, B. tabaci, Myzus persicae (peach-potato aphid) and Ctenocephalides felis (cat flea) from areas of intensive neonicotinoid use for phenotypic characterisation of resistance and molecular characterisation of possible target-site resistance mechanisms. The latter will include membrane binding studies and sequencing of nAChR genes to detect mutations putatively associated with reduced binding, and expression studies to investigate such an association. Based on these findings we will develop and validate high throughput DNA-based assays for diagnosing resistance mutations in individual insects in order to monitor their incidence and aid resistance management strategies.

The neonicotinoids are a highly successful class of insect-selective nicotinic acetylcholine receptor (nAChR) agonists that are now used extensively for insect pest control in crop protection and animal health. Their widespread use has however led to reports of resistance in several important pest species. In this study, we will exploit recent work on the molecular analysis of nAChR subunit genes and our involvement in key resistance monitoring networks to evaluate the current status of resistance to these compounds in key target species including planthoppers, aphids, whiteflies and fleas, and to analyse resistant strains for possible target site mechanisms and mutations. This will involve membrane binding studies with nicotinic radioligands to look for alterations in the affinity of the target, coupled with a detailed molecular (sequence) characterisation of the individual nAChR subunit genes to identify the mutations that may be associated with reduced binding and hence resistance. Any such mutations, together with a single amino acid substitution already identified in brown planthoppers (Y151S), will then be subjected to more detailed examination of their effects on ligand binding through the expression and analysis of wild-type and mutated receptor subunits in Drosophila S2 cells and Xenopus oocytes. In addition to identifying mutations that can arise to confer resistance, we anticipate that these studies will also generate novel insights into the key ligand-receptor interactions that influence the potency and insect-selectively of this important class of insecticides at the nAChR. Finally, we will develop and validate high throughput DNA-based assays for diagnosing resistance mutations in individual insects in order to monitor their incidence in field populations and thereby aid resistance management strategies. (joint with Denholm BB/E010601/1)