A genomic approach to understanding insecticide resistance in crop pests

Insect pests of crops are often controlled using chemical insecticides. Unfortunately over time many pests have evolved resistance to the insecticides used for control. Insects have been shown to develop resistance in two main ways. Firstly by changes in the protein that the insecticide binds to which means that it is no longer as sensitive to the toxic effect of the insecticide and secondly by increased production of enzymes that break down or bind to the insecticide and render it ineffective. In this proposal we aim to study insecticide resistance in two important crop pests the peach potato aphid (Myzus persicae) and the brown planthopper (Nilaparvata lugens). M. persicae is a major pest on a range of crops in the UK and Europe and N. lugens is a major pest of rice crops in Asia, both cause damage to plants through direct feeding and the transmission of viruses resulting in high economic losses. Both of these crop pests have evolved resistance to many of the insecticides used for their control and the main chemical class currently being used is the neonicotinoids. However reports of resistance to this insecticide class have recently been described. Biochemical studies have shown that this resistance is likely to be caused by increased production of enzymes that break down the insecticide, in particular a group of enzymes called cytochrome P450 monooxgenases (P450s). P450s are a class of enzymes with many functions including the breakdown of toxins and insects have been found to have between 46-143 P450 genes, each producing a different enzyme. Insect pests can become resistant to insecticides by increasing the amount of one or more of the P450 enzymes they produce. In this project we aim to examine if resistance in M. persicae and N. lugens to neonicotinoids is caused by over-production of P450s and determine which P450s are involved and why they are over-produced. It is not easy to study the large gene families involved in metabolic resistance however recent advances in the field of genomics (the study of genes and their function) and new associated technologies means that it is now more feasible. This study will exploit these new resources to identify P450 genes in the target pest species. These include the genome (the entire DNA content of an organisms) sequences of a number of insect species (including an aphid), expressed sequence tags or ESTs (small pieces of DNA sequence usually 200 to 500 nucleotides long that are generated by sequencing either one or both ends of an expressed gene) and affordable high-throughput sequencing technologies that allow many hundreds of millions of bases (a unit of DNA) of sequence to be determined in a matter of hours. The identified P450 genes will then be studied using new molecular methods that allow determination of the levels of expression of genes into RNA and protein. The technique RNA interference (the introduction of double-stranded RNA into a cell to inhibit the expression of a gene) will be used to silence P450 genes and therefore examine their role in resistance. Finally the P450 genes will be cloned and expressed as protein to see if they break down or bind to insecticide. When the specific P450s involved in resistance in these crop pests have been identified we will develop diagnostic tools to monitor insect populations for resistance. These are an essential requirement of resistance management strategies which aim to slow or prevent the development of resistance. Prolonging the life of insecticides by managing resistance is vital as there are only a limited number of insecticides available for control and proposed new legislation on pesticides from the European Parliament will dramatically cut the availability of insecticides for use in agriculture. This project will be carried out in collaboration with partners in agrochemical companies and the Insecticide Resistance Action Group to ensure the findings of this study can be rapidly exploited.

Insecticide resistance in crop pests has been an ongoing problem since the introduction of synthetic insecticides in the 1940's and has been shown to develop through two main mechanisms 1) the increased production of enzymes which can break down or bind to the insecticide and 2) structural changes in the target protein that render it less sensitive to the toxic effects of the insecticide. In this proposal we aim to study insecticide resistance in two important crop pests the peach potato aphid (Myzus persicae) and the brown planthopper (Nilaparvata lugens). M. persicae is a major pest on a range of crops in the UK and Europe and N. lugens is a major pest of rice crops in Asia, both cause damage to plants through direct feeding and the transmission of viruses resulting in high economic losses. The main insecticide class currently used to control M. persicae and N. lugens are the neonicotinoids, however reports of resistance to this insecticide class have recently been described and there is strong evidence that elevated cytochrome P450 monooxgenases may be a contributing cause. In the proposed study we will explore the role that P450s play in metabolic resistance to neonicotinoids in the two pest species. We will identify P450 genes in M. persicae and N. lugens by exploiting genomic resources and new associated technologies that have recently become available including >24 insect genome sequences (including the recently sequenced pea aphid Acyrthosiphon pisum genome), EST databases and affordable high-throughput sequencing technologies. Identified P450s will then be studied using state-of-the art post-genomic technologies including quantitative PCR, microarrays, RNAi and functional expression to identify which enzymes are involved in resistance and why they are over-expressed. The findings will be exploited to develop diagnostic tools to monitor populations for resistance as part of management strategies which aim to slow or prevent the development of resistance.