Sims, Cassie Marie
(2020)
Elucidation of insect olfaction in the pea aphid, Acyrthosiphon pisum.
PhD thesis, University of Nottingham.
Abstract
Aphids (Homoptera; Aphididae) are major pests on arable and horticultural crops. Aphids communicate using olfaction, employing semiochemicals such as the alarm pheromone, (E)-β-farnesene, and the sex pheromone components, (4aS,7S,7aR)-nepetalactone and (1R,4aS,7S,7aR)-nepetalactol. An understanding of olfaction in aphids, specifically molecular recognition, and discrimination of odorants, can help when designing novel protection tools. However, little is understood about the aphid olfactory system, specifically, the role of odorant-binding proteins (OBPs), small, soluble proteins that are found in high concentrations in sensory organs (antennae). In this thesis, the molecular recognition of the sex pheromone in the pea aphid, Acyrthosiphon pisum was investigated by exploring the roles of olfactory proteins such as OBPs and odorant receptors (ORs)
Initially, aphid sex pheromone components, (4aS,7S,7aR)-nepetalactone and (1R,4aS,7S,7aR)-nepetalactol, and their respective non-naturally occurring enantiomers, (4aR,7R,7aS)-nepetalactone and (1S,4aR,7R,7aS)-nepetalactol were synthesised, with the synthesis optimised at various points, giving a final yield of approximately 1%. A. pisum OBPs were also expressed on a litre-scale and purified in large quantities (> 1mg). Binding activity between A. pisum OBPs and sex pheromone components was screened using in silico methods. These screens revealed OBP6 as a strong candidate for binding of the sex pheromone components due to low KDs, and OBP9 as a protein with little binding activity with the ligands of interest. Furthermore, potential strong-binding analogues of nepetalactol and nepetalactone were predicted through in silico methods. These analogues have the potential to act as olfactory disrupters and may be a valuable pest control method in future. In silico work also predicted the binding sites for electrophysiologically active molecules in ORs, whilst predicting the potential sex pheromone binding OR in aphids.
After successful in silico screens, a range of fluorescence binding assays were performed with OBP6 and OBP9 and the sex pheromone components. The fluorescence assays used a non-traditional approach, observing intrinsic fluorescence of the tryptophan residues both alone and in the presence a range of fluorescent probes. These assays confirmed the predicted results of the in silico screens. OBP6 was seen to bind sex pheromone components, with a particularly low KD of 1.30 ± 0.60 µM observed for the interaction between OBP6 and (4aS,7S,7aR)-nepetalactone. Due to OBP6’s high expression in the aphid antennae, these results suggest it may play a role in sex pheromone perception.
To further explore the interaction between OBPs and the sex pheromone components, other methods, including mass spectrometry, nuclear magnetic resonance (NMR) and gas-chromatography (GC), were employed. Mass spectrometry experiments were unsuccessful, but Saturation Transfer Difference (STD) NMR and a biphasic GC assay both gave binding results for OBP6 consistent with the in silico and fluorescence experiments. The STD-NMR assay also allowed for epitope mapping of the binding interactions between the sex pheromone components and OBP6, which aligned with the predicted conformations from in silico screens.
Overall, OBP6 was shown to bind the sex pheromone component (4aS,7S,7aR)-nepetalactone with a higher affinity than other ligands, including the alarm pheromone (E)-β-farnesene. The results of this work suggest a role for OBP6 in sex pheromone binding. All assays focused on the enantiomeric discrimination potential of OBP6. Though no clear discrimination was observed in this study, levels of discrimination could be seen in the fluorescence assays. Further work is required to fully elucidate OBP6’s potential role.
Future work will focus on the exploration of OBP6’s role in aphid olfaction, from behavioural studies to further binding assays and structural determination. Additionally, results from this thesis may be adapted for use in the development of novel pest control tools.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Oldham, Neil
Birkett, Michael A.
Stockman, Robert
Withall, David |
| Keywords: |
insect, olfaction, odorant-binding proteins, OBPs, aphid, aphids, nepetalactone, nepetalactol, sex pheromone |
| Faculties/Schools: |
UK Campuses > Faculty of Science > School of Chemistry |
| Item ID: |
63918 |
| Depositing User: |
Sims, Cassie
|
| Date Deposited: |
06 Jun 2023 08:42 |
| Last Modified: |
06 Jun 2023 08:44 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/63918 |
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