Molecular analysis of olfactory perception in Drosophila melanogaster

Abstract

Over the last decade the insect olfactory system has emerged as an important model system with which to investigate the biochemical basis of eukaryote signalling processes. It is believed that certain odorant degrading enzymes are required to maintain the ongoing sensitivity of an insect’s olfactory neuronal system by repriming neurons. However, relatively few ODEs have been identified and characterized to date, especially in the model insect Drosophila melanogaster. The study presented here takes biochemical, neurobiological and behavioural approaches to elucidate the role of ODEs in D. melanogaster. After a review of relevant literature in Chapter 1, Chapter 2 decodes the antennal transcriptome of D. melanogaster for the first time. Using high quality genome sequence and transcriptomic data for many other tissues of this species already available, I identified a few antennae-selective esterases, cytochrome P450s (P450s), glutathione S-transferases (GSTs) and UDP-glycosyltransferases (UGTs). Of these, the activity of one esterase JHEdup, against a range of volatile odorants was found to be comparable with other known ODEs from different species, mainly Lepidoptera. I also identified the presence of another esterase, EST6, at high levels in the antennae. It has previously been proposed that EST6 is a catalyst for the transformation of pheromonal and kairomonal esters to the corresponding alcohols and acids, thereby mediating various mating behaviours. I further examined the proposed effect of EST6 by comparing wild type and EST6 null flies at a neurobiological and behavioural level. The findings, presented in Chapter 3, show this enzyme is important for the flies to respond to incoming volatile odorants and affects their subsequent behaviours. Additionally, EST6 has previously been reported to hydrolyse cis-vaccenyl acetate (cVA), the major pheromone known in Drosophila, in vitro and recent electroantennogram (EAG) experiments with EST6 wild type and null flies exposed to cVA suggest that this might also be an in vivo function. I therefore conducted experiments to understand the biochemical activity of EST6 against cVA. I also measured its activity against 84 other bioactive esters. The results categorically show that EST6 has no activity against cVA but has very good activity against a wide range of fruit- and yeast-derived volatiles known to play a role in mediating female reproductive behaviour. These results are presented in Chapter 4, along with a crystal structure of EST6. The final chapter of this thesis then discusses the overall findings of these studies and offers a broader perspective on future directions. The three major conclusions from the work are as follows. Firstly, JHEdup and EST6 are broad range ODEs active against a wide range of food odorants. Secondly, EST6 may also have a role in cVA processing but not actually as an ODE against this substrate. Thirdly, ODEs may be a fruitful system to develop biocontrol systems for pest insects based on disrupting their olfactory system.