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Title: A genomic approach to understanding metabolic insecticide resistance to diamides in diamondback moth (Plutella xylostella)
Author: Mallott, M.
ISNI:       0000 0004 6425 283X
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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The diamondback moth (Plutella xylostella) is a destructive insect pest of cruciferous plants throughout the world. Control of diamondback moth relies heavily on the use of chemical insecticides, resulting in this species developing resistance to most compounds. The diamides are a relatively recently introduced insecticide class used to control P. xylostella. Mutations in the diamide target site that confer resistance to diamides have recently been described, however at least one strain exists without these mutations, but remains resistant to diamides. This study exploited genomic resources including whole genome microarrays to identify candidate detoxification genes. One candidate, a flavin-dependent monooxygenase (FMO2) showed approximately a 70-fold increase in expression in microarrays and 14,700-fold overexpression in qPCR when compared to the susceptible ROTH strain. This and other candidate genes were functionally analysed using multiple approaches. In the absence of diamide selection the resistant Hawaii selected (HS) strain of P. xylostella rapidly lost resistance, and this was associated with a drop in the expression of FMO2. Transgenic D. melanogaster expressing P. xylostella FMO2 exhibited significant resistance to chlorantraniliprole suggesting it has the capacity to detoxify this compound. In contrast, transgenic D. melanogaster expressing other candidate detoxification genes were not resistant. Insecticide bioassays of both P. xylostella and transgenic Drosophila (over)expressing FMO2 revealed significant differences in sensitivity to anthranilic and phthalic diamides, suggesting FMO2 can metabolise the former but not the latter. Liquid chromatography-mass spectrometry analysisfollowing diamide exposure showed significant differences in chlorantraniliprole stability in the HS P. xylostella and FMO2 transgenic D. melanogaster strains compared to their susceptible counterparts, implying enhanced metabolism and or excretion may be involved in resistance. The putative promoter region of the FMO2 gene in the resistant and susceptible P. xylostella strains was found to contain numerous SNPs and indels. These may have in part, resulted from a transposable element insertion in the promoter of the resistant strain, leading to modification in the number and type of predicted transcription factor binding sites. In a dual luciferase reporter assay, the HS promoter variant drove significantly higher levels of expression than the ROTH promoter suggesting a cis-acting element is, at least in part, responsible for the overexpression of FMO2 in the resistant strain. In summary, this PhD provides lines of evidence for a novel mechanism of insecticide resistance based on the overexpression of a FMO2. The importance of this enzyme family in humans has been well established, the results detailed in this PhD suggest they may also play a role in xenobiotic detoxification in insects. Finally, as diamides are a relatively new and important means of controlling P. xylostella the findings presented here hold potential to contribute to the future control of this economically important species.
Supervisor: Paine, M. ; Wondji, C. ; Bass, C. ; Field, L. ; Elias, J. ; Slater, R. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral