Drosophila, metabolomics and insecticide action

The growing problem of insecticide resistance is jeopardising current pest control strategies and current insecticide development pipelines are failing to provide new alternatives quickly enough. Metabolomics offers a potential solution to the bottleneck in insecticide target discovery. As a proof of concept, metabolomics data for permethrin exposed Drosophila melanogaster was analysed and interpreted. Changes in the metabolism of amino acids, glycogen, glycolysis, energy, nitrogen, NAD+, purine, pyrimidine, lipids and carnitine were observed along with markers for acidosis, ammonia stress, oxidative stress and detoxification responses. Many of the changed metabolites and pathways had never been linked to permethrin exposure before. A model for the interaction of the observed changes in metabolites was proposed. From the metabolic pathways with the largest changes, candidate genes from tryptophan catabolism were selected to determine if the perturbed pathways had an effect on survival when exposed to permethrin. Using QPCR it was found that all genes in the entire pathway were downregulated by permethrin exposure with the exception of vermilion suggesting an active response to try and limit flux through tryptophan catabolism during permethrin exposure. Knockdown of the tryptophan catabolising genes vermilion, cinnabar and CG6950 in Drosophila using whole fly RNAi resulted in changes in susceptibility to permethrin for both topical and oral routes of exposure. Knockdown of the candidate genes also caused changes in susceptibility when the insecticides fenvalerate, DDT, chlorpyriphos and hydramethylnon were orally administered. These results show that tryptophan catabolism knockdown has an effect on surviving insecticides with a broad range in mode of action. Symptoms that occur in Drosophila during exposure to the different insecticides were also noted. To gain further understanding into the mechanisms affecting survival, tissue specific knockdown was performed revealing tissue and gender specific changes in survival when vermilion, cinnabar and CG6950 are knocked down. Metabolomics was performed on the knockdown strains to determine the efficacy of the knockdowns on tryptophan catabolism and to identify any knock-on effects. The results indicate that tryptophan metabolite induced perturbations to energy metabolism and glycosylation also occur in Drosophila along with apparent changes in the absorption of ectometabolites. As the knockdown of vermilion, cinnabar and CG6950 tended to result in reduced susceptibility to insecticides, they would make poor targets for insecticidal compounds, however, they may be the first examples of genes that are not directly involved in insecticide metabolism or cuticle synthesis that increase insecticide tolerance in Drosophila. As the first metabolomics data set showed evidence for oxidative stress during permethrin exposure, preliminary work was begun for identifying the tissue specificity and timing of oxidative stress in both Dipterans and Lepidopterans using Drosophila and Bombyx mori as models. In Drosophila oxidative stress did not begin immediately suggesting that the insecticide itself is not a cause, however, a rapid increase in oxidative stress occured over a six hour period after a day of oral exposure implicating catabolites of permethrin. Bombyx were highly susceptible to permethrin showing oxidative stress in the Malpighian tubule and silk gland when exposed. This study has shown that metabolomics is highly effective at identifying pathways which modulate survival to insecticide exposure. It has also brought insight into how insecticide induced pathology may cause death. Data has also been generated which could help characterize the putative transaminase CG6950