Current Research Projects
Exploring behavioural impacts of insecticides and roles of their receptor targets
Recent work has identified a number of the nicotinic acetylcholine receptor (nAChRs) subunits as targets of widely applied commercial insecticides. These insecticides act as agonists or allosteric agonists that lead to death of the insect at field applied rates of insecticide through knockdown and paralysis of the insect due to over stimulation of these receptors in nervous system. One of the insecticide classes known to target nAChRs, the neonicotinoids, has been associated with detrimental effects on survival of bees and other beneficial insects which has led to some countries banning its use. Some of the receptor subunits involved in the mode of action of the neonicotinoids are already known to have roles in behaviours such as courtship and sleep patterns . Many nAChR genes are expressed in the mushroom body, a major sensory and behavioural processing centre of the brain linked to learning and memory  and odorant response . The aims of this project are to understand which of the nicotinic acetylcholine receptors contribute to different types of learning and memory in insects and assess the likely fitness costs of insecticide exposure or resistance mutations on insects. This will be considered both in terms of development of field resistance and as a threat to beneficial insect survival.
Approaches used to address these questions will be:
- Perform learning and memory paradigms on insecticide exposed and nAChR mutant flies. Odorant response will be assessed using larval attraction and adult olfactory trap apparatus . Non-associative learning tests will use olfactory or light-off jump reflexes so habituation or sensitisation can be determined.
- Create strains to conduct somatic CRISPR/Cas9 knockout experiments specifically in sets of specific neurons.
- Confirm the importance of receptor expression in the mushroom body through specific somatic CRISPR/Cas9 knockout of nAChR subunits in this and other regions of the brain.
1. Somers, J., H. N. Luong, J. Mitchell, P. Batterham, and T. Perry. “Pleiotropic Effects of Loss of the Dalpha1 Subunit in Drosophila Melanogaster: Implications for Insecticide Resistance.” Genetics 205, no. 1 (Jan 2017): 263-71.2016
2. Vogt K, Schnaitmann C, Dylla KV, Knapek S, Aso Y, Rubin GM, Tanimoto H. “Shared mushroom body circuits underlie visual and olfactory memories in Drosophila.” Elife. 2014 Aug 19;3:e02395. doi: 10.7554/eLife.02395.
3. Guven-Ozkan T, Davis RL. “Functional neuroanatomy of Drosophilaolfactory memory formation.” Learning & Memory. 2014;21(10):519-526. doi:10.1101/lm.034363.114.
4. C Woodard, T Huang, H Sun, S L Helfand and J Carlson. “Genetic analysis of olfactory behavior in Drosophila: a new screen yields the ota” GENETICS October 1, 1989 vol. 123(2) 315-326.
Manipulating nicotinic acetylcholine receptor structure to examine native receptor compositions and identify functional domains
The nicotinic acetylcholine receptors translate a chemical signal into an electrical signal through the binding of their ligand, acetylcholine, that leads to a conformational shift allowing the influx of cations through their membrane spanning pore into the cell. These receptors are insecticide targets and a large proportion of research has been focussed towards increasing our understanding of how and where various insecticides bind at the N-terminal ligand binding domain. What has not been established is which specific receptor subunit combinations are formed in vivo. The subunit composition of a pentameric nAChR will dictate its pharmacological properties such as ligand affinity, ion permeability and gating kinetics. While work in our lab has examined the roles individual receptor subunits have in response to different insecticides, this sensitive phenotypic readout of receptor function has not been sufficient to define the specific receptor subtypes found in vivo.
To do this, the receptor subunit order of assembly into the pentameric complex needs to be controlled through specific concatenation of receptor subunits which will then allow the examination of particular receptor subtypes in a controlled background. We can then assess their response to ligands and impacts on other behavioural phenotypes. Further work to define the protein domains of nAChR subunits involved in binding of compounds and conferring particular pharmacological properties to receptor subtypes is also possible given our knowledge of which subunits bind different insecticides. Using our recently published GAL4>UAS rescue systems [5,6], chimeric proteins can be expressed to allow us to probe the receptor structure for key functional domains . Comparison of phenotypic results will be made to homology models of the receptor interfaces and interacting domains, providing evidence of these as accurate models of native receptors.
Approaches used to address these questions will be to:
Use molecular techniques to create chimeric and concatenated nAChR subunit constructs and to transform melanogaster with these.
- Create strains and conduct somatic CRISPR/Cas9 crossing that will specifically express these constructs in appropriate knockout backgrounds.
- Validation of the function of these constructs using insecticide bioassays and/or behavioural assays.
5. Perry, T., J. Somers, Y. T. Yang, and P. Batterham. “Expression of Insect Alpha6-Like Nicotinic Acetylcholine Receptors in Drosophila Melanogaster Highlights a High Level of Conservation of the Receptor:Spinosyn Interaction.” Insect Biochem Mol Biol 64 (Sep 2015): 106-15.
6. Somers, J., H. N. Luong, J. Mitchell, P. Batterham, and T. Perry. “Pleiotropic Effects of Loss of the Dalpha1 Subunit in Drosophila Melanogaster: Implications for Insecticide Resistance.” Genetics 205, no. 1 (Jan 2017): 263-71.2016
7. Somers, J., J. Nguyen, C. Lumb, P. Batterham, and T. Perry. “In Vivo Functional Analysis of the Drosophila Melanogaster Nicotinic Acetylcholine Receptor Dalpha6 Using the Insecticide Spinosad.” Insect Biochem Mol Biol 64 (Sep 2015): 116-27.
Assessing the importance of accessory proteins on nicotinic acetylcholine receptor function
There has been a bias in research of insect nicotinic acetylcholine receptors towards the interaction of receptors with their ligands which has meant many other areas of nicotinic acetylcholine receptor biology remain poorly understood. Very little is known about important stages in the production of functional receptors prior to their localisation to the synapse. A nAChR subunit needs to be folded correctly, assembled into its correct pentameric conformation and then trafficked to the membrane. Maintaining the appropriate synaptic localisation and modulation of receptor pharmacokinetic properties are further examples of the extensive roles accessory proteins play in regulating these receptors at the synapse. Several proteins have recently been associated with impacts on nAChR function including sss/Lynx, nacho and Dmric-3, while many others are yet to be identified [8-10]. Using fluorescently tagged receptor subunits and somatic CRISPR/Cas9, this project aims to characterise specific interactions of these proteins with individual nAChR subunits and determine their in vivo effects on receptor protein levels, responsiveness and localisation.
Approaches used to address these questions will be to:
Create strains and conduct somatic CRISPR/Cas9 crossing that will specifically knockout accessory proteins individually and in combination.
- Visualise impacts of the loss of accessory protein on receptor localisation and levels using confocal microscopy and fluorescently tagged receptor subunits
- Validation of the impact of loss of accessory protein function using insecticide bioassays and/or behavioural assays.
8. Miwa JM, Stevens TR, King SL, Caldarone BJ, Ibanez-Tallon I, Xiao C, Fitzsimonds RM, Pavlides C, Lester HA, Picciotto MR, Heintz N. “The prototoxin lynx1 acts on nicotinic acetylcholine receptors to balance neuronal activity and survival in vivo.” Neuron. 2006 Sep 7;51(5):587-600.
9. Boulin T, Gielen M, Richmond JE, Williams DC, Paoletti P, Bessereau JL. “Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor.” Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18590-5. doi: 10.1073/pnas.0806933105. Epub 2008 Nov 19.
10. Gu S, Matta JA, Lord B, Harrington AW, Sutton SW, Davini WB, Bredt DS. “Brain α7 Nicotinic Acetylcholine Receptor Assembly Requires NACHO.” Neuron. 2016 Mar 2;89(5):948-55. doi: 10.1016/j.neuron.2016.01.018. Epub 2016 Feb 11.