Drosophila melanogaster as a model to study insecticide mode of action

Abstract

The development of novel insecticides requires insect models to determine mode of action. This project aimed to assess Drosophila as a model system to study insecticide mode of action. Drosophila larvae were used to confirm the action of the spiroindoline insecticide 5Cl-CASPP (CASPP). Experiments presented confirm that this compound inhibits acetylcholine (ACh) loading into synaptic vesicles (SVs) by inhibiting the vesicular acetylcholine transporter (VAChT). Mortality assays showed that CASPP induced lethality is reduced by either overexpression of VAChT or expression of a resistant transporter (VAChTY49N). Larval tracking demonstrated a sub-lethal effect of CASPP on cholinergic-regulated locomotion. Whole-cell patch recordings from identified motoneurons, that receive excitatory cholinergic synaptic input, showed reduced frequency of release of ACh-containing SVs (i.e. minis) following exposure to CASPP. Mini amplitude was unaffected. By contrast, overexpression of VAChT resulted in a marked increase in frequency of minis but again no change to amplitude. Expression of VAChTG342R that, in C.elegans, is unable to bind synaptobrevin does not affect either mini frequency or amplitude. This suggests that VAChT may also govern ACh release, in addition to SV loading.While overexpression of wild-type VAChT increases mini frequency and not amplitude, increases to both were observed following expression of a VAChT variant with a polymorphism (missing glutamine; Q) in a unique polyQ domain. Ultrastructural examination of the active zone found that changes to SV release were not accompanied by changes to active zone morphology, SV size or number. This was supported by the finding of no significant difference in total SVs released in the -Q VAChT variant when SV recycling was blocked. Taken together, the data are indicative that individual SVs are filled to a greater level following expression of this VAChT variant. This identifies the polyQ domain as a potential regulator of SV loading and, moreover, may provide an attractive target for novel insecticide development.This project also assessed the Giant Fiber System (GFS) in adult Drosophila as a potential medium throughput model to rapidly identify insecticide mode of action. The GFS is activated by electrical stimulation and output measured as excitatory junctional potentials (EJPs) in leg and flight muscles. Flies were treated with compounds of different known modes of action (cholinergic, glutamatergic etc.) in order to characterise effect. Novel compounds with unknown modes of action were also tested and effects compared. This system is suitable to indicate insecticides with a cholinergic mode of action but requires additional characterisation to fully understand the relationship between mode of action of compounds and GFS output.

Date of Award	1 Aug 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Catherine Mccrohan (Supervisor) & Richard Baines (Supervisor)