Abstract
Global agriculture and the control of insect disease vectors have developed with a heavy reliance on insecticides. The increasing incidence of resistance, for virtually all insecticides, threatens both food supply and effective control of insect borne disease. CASPP ((5-chloro-1’-[(E)-3-(4-chlorophenyl)allyl]spiro[indoline-3,4’-piperidine]-1-yl}-(2-chloro-4-pyridyl)methanone)) compounds are a potential new class of neuroactive insecticide specifically targeting the Vesicular Acetylcholine Transporter (VAChT). Resistance to CASPP, under laboratory conditions, has been reported following either up-regulation of wildtype VAChT expression or the presence of a specific point mutation (VAChTY49N). However, the underlying mechanism of CASPP-resistance, together with the consequence to insect viability of achieving resistance, is unknown. In this study, we use electrophysiological characterisation of cholinergic release at Drosophila larval interneuron→motoneuron synapses to investigate the physiological implications of these two identified modes of CASPP resistance. We show that both VAChT up-regulation or the expression of VAChTY49N increases miniature (mini) release frequency. Mini frequency appears deterministic of CASPP activity. However, maintenance of SV release is not indicative of resistance in all cases. This is evidenced through expression of syntaxin or complexin mutants (sytx3-61/cpxSH1) that show similarly high mini release frequency but are not resistant to CASPP. The VAChTY49N mutation additionally disrupts action potential-evoked cholinergic release and fictive locomotor patterning through depletion of releasable synaptic vesicles. This observation suggests a functional trade-off for this point mutation, which is not seen when wildtype VAChT is up-regulated.
Highlights
Vesicular transporters load neurotransmitter into synaptic vesicles (SV) for storage before release
To validate the previously published observation of 5-Cl-CASPP resistance when Vesicular Acetylcholine Transporter (VAChT) is up-regulated, and to ascertain resistance associated with endogenously expressed VAChT Y49N, mortality assays were conducted in larvae where VAChT was up-regulated in all cholinergic neurons or expression of VAChT Y49N achieved through CRISPR knock-in (VAChT Y49N/Y49N and VAChT Y49N/+)
We show in this study that resistance may be attained through VAChT up-regulation, adult longevity is reduced
Summary
Vesicular transporters load neurotransmitter into synaptic vesicles (SV) for storage before release. Transporter localisation dictates loading substrate and differs between vesicle classification [1]. Small clear SVs mostly store fast-acting neurotransmitters. VAChTY49N perturbs synaptic transmission any additional role in data collection and analysis or decision to publish, The specific roles of these authors are articulated in the ‘author contributions’ section
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
