The Role Of Post-Transcriptional Modifications Of Nicotinic Acetylcholine Receptor Subunits On The Toxicity Of Spinosad And Imidacloprid
Spinosad and imidacloprid are two of the most widely used insecticides. Both of these compounds act at the nicotinic acetylcholine receptor through mechanisms unique to each insecticide. High levels of resistance have been reported from a number of important agricultural and economic pests across the globe, often within a few years after the introduction of these insecticides. Studies with laboratory created strains of Drosophila melanogaster indicate spinosad targets nicotinic acetylcholine receptors that contain the D[alpha]6 subunit. In an effort to validate these laboratory findings, I sequenced the Pxyl[alpha]6 subunit from the field collected Pearl-Sel strain of diamondback moth, Plutella xylostella, which has more than 18,000-fold resistance to spinosad. The Pxyl[alpha]6 subunit in Pearl-Sel possesses numerous premature stop codons that are unseen in two other spinosad susceptible strains. These truncated transcripts are genetically associated with spinosad resistance through the use of the F2 backcross-bioassay method. I chose to utilize RNAi in the red flour beetle, Tribolium castaneum, to systematically investigate the role of other nicotinic acetylcholine receptor subunits on toxicity of spinosad because RNAi is very robust in this species. I cloned of all 12 nAChR subunits in T. castaneum to use as templates for the production of dsRNA to use in RNAi. Sequencing these transcripts revealed a diverse array of posttranscriptional modifications such as alternative and cassette exon use, intron retention, intron 3′ splice site variations, and a vast number of alleles. I used this information to design effective RNAi for the Tcas[alpha]6 because my work on P. xylostella, and other work on D. melanogaster indicate that [alpha]6 null mutants are resistant to spinosad. RNAi was induced by injecting double stranded RNA for Tcas[alpha]6 into pupae of T. castaneum. Silencing of Tcas[alpha]6 produced no change in spinosad LC50 values despite a reduction in the expression of Tcas[alpha]6. To confirm this result, RNAi against the D[alpha]6 subunit of D. melanogaster was performed using the Gal4-UAS system. There was no change in spinosad sensitivity in flies due to D[alpha]6 silencing despite a significant reduction in D[alpha]6 expression. These results indicate that RNAi against nicotinic acetylcholine receptors is not a feasible system to study the effect of specific subunits on insecticide sensitivity due to the large differences in the expression of nicotinic acetylcholine receptors and the RNAi machinery. The Gal4-UAS system was utilized to silence the expression of Adenosine Deaminase Acting on RNA (ADAR) in different tissues of D. melanogaster. I chose this approach because it has been demonstrated that the Gal4-UAS system is effective at reducing the expression of ADAR, the level of ADAR expression is similar to the expression level of the RNAi machinery, and A-to-I RNA editing may be a factor in insecticide resistance. These ADAR-deficient flies were subject to spinosad and imidacloprid bioassays. Ubiquitous reduction in ADAR resulted in decreased spinosad insensitivity, while reduction in ADAR in cholinergic neurons and muscle increased spinosad insensitivity. Reduction of ADAR expression in cholinergic neurons, muscle, and glia increased imidacloprid insensitivity. These results indicate that editing is an important factor in insecticide insensitivity and the effect of editing is not spatially homogenous in the fly. I used the peak height ratio method to estimate the frequency of A-to-I RNA editing to ensure the rate of editing was reduced via the Gal4-UAS system. The use of an antisense primer showed very accurate and precise measurements of A-to-I RNA editing based on known editing rates. The accuracy and precision was consistent across different editing sites and expected editing frequencies. This method is more cost and time effective in comparison to other contemporary methods. These results provide valuable insight into understanding and managing insecticide resistance. Firstly, they validate the use of a model organism to predict resistance in the instance of spinosad resistance. Secondly, they suggest that RNAi of nAChRs is not a suitable technique to evaluate target sites of spinosad and imidacloprid. Thirdly, A-to-I RNA editing affects the toxicity of spinosad and imidacloprid that varies depending on the tissues where it is expressed. These results will be of utmost importance in studies on population genetics, physiology, neurobiology, and mechanisms of insecticide resistance.
insecticide; nicotinic acetylcholine receptor; resistance
Scott, Jeffrey Graham
Harris-Warrick, Ronald Morgan; Gilbert, Cole
Ph.D. of Entomology
Doctor of Philosophy
dissertation or thesis