The genetic basis for resistance to bioinsecticides in diamondback moth Plutella xylostella

Recent years have seen the development of several bio-insecticides, which are often very specific in their mode of action and therefore potentially more environmentally friendly than traditional chemical insecticides. It was even suggested that, due to their biological origin, these insecticides might avoid the evolution of resistance in targeted pest species. Unsurprisingly this has not proven to be the case, and increased agricultural use of two of these insecticides, Bt and Spinosad, has led to the evolution of resistance in the diamondback moth, the most destructive global pest of cruciferous crops. Understanding the mode of action and mechanisms of resistance is crucial for long term, sustainable use of these compounds. Furthermore, although transgenic Bt crops are not available for commercial use in the UK, an understanding of the basis for resistance to existing Bt crop sprays will be vital in assessing the likelihood of resistance to transgenic crops in the future. Surprisingly, despite its global importance in both foliar sprays and transgenic crops, the targets for Bt toxin are far from clear. Here, we aim to characterise the genetic basis of resistance in populations of diamondback moth that have acquired resistance to these insecticides in the field. Both Bt (Cry1Ac toxin) and spinosad resistance are known to involve a single major gene and in both cases we have already identified linked molecular markers. We will target the regions of the genome surrounding these resistance genes with anonymous molecular markers. Then, by means of a large-insert BAC library, we will fully sequence the region surrounding the two resistance genes. In order to identify the resistance gene we will then test candidate genes identified from this region to see whether they are expressed in the larvae and in appropriate tissues such as the midgut, and also whether there are differences in expression between resistant and susceptible populations. Additionally, we will use RNAi knockdown experiments to determine whether there is an effect of reducing the expression of the candidate gene on levels of resistance in susceptible lines. We will also characterise sequence differences, if any, between the resistant and susceptible lines in the gene transcript of the candidate locus. This will allow development of rapid PCR-based methods for detecting resistant alleles in field caught samples that will facilitate field monitoring of the spread of resistance alleles. Identification of insecticides with novel modes of action can play an important role in preventing the evolution of cross resistance in populations of agricultural pests, and novel biopesticides such as Bt and spinosad have played an important role in complementing and/or replacing older chemical insecticides. However the evolution of resistance is inevitable in targeted pests unless measures are taken to combat it. A better understanding of the mechanisms of resistance, and hence the mechanism of action of these compounds will be crucially important in developing novel toxins with distinct modes of action, and in developing strategies for combating resistance in the field.

Bt and Spinosad are two widely used, biologically derived insecticides. Only one pest species, the diamondback moth, Plutella xylostella, has developed field resistance to both compounds. Diamondback moth strains with monogenic resistance to Spinosad and Bt (Cry1Ac) toxins have already been characterised. Here we will identify the genetic basis of resistance in these strains. By taking a forward genetics approach to identification of the genes, we will not be relying on any prior identification of candidate loci, although we will obviously take advantage of previous work in suggesting the most likely genes as we uncover sequence information for the relevant genomic region. Backcross broods will be reared that segregate for resistance, 80% of which will be treated with the respective insecticide toxin. A dual AFLP approach will be used, based on both genomic and cDNA templates, which will identify markers associated with susceptible alleles (these will be absent from the treated brood offspring). These AFLP markers will then be used to screen a large insert genomic BAC library, and a BAC contig assembled around each of the two resistance loci. Six clones will be identified and sequenced to identify all ORFs in the region of the resistance loci. Various methods will be used to confirm the identity of the resistance locus from among the ORFs identified from the BAC sequences. First, RT-PCR of cDNA from larval midgut of susceptible and resistant strains will confirm a) whether the gene is expressed in the relevant tissue and b) whether there is any evidence for differential expression between susceptible and resistant strains. Second, characterisation of gene transcripts from susceptible and resistant strains will identify any coding sequence differences between the alleles associated with the two phenotypes and third, RNAi knockdown experiments will then provide an experimental test of the role of candidate loci in resistance.