Genetics - Theses

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    Nicotinic acetylcholine receptors; an examination of expression and insecticide interactions in Drosophila melanogaster
    ALI, SHAHID ( 2012)
    Nicotinic acetylcholine receptors (nAChRs) are complex transmembrane proteins that belong to the Ligand-gated ion-channel (LGIC) super-family. They are responsible for cholinergic synaptic transmission in the central nervous system (CNS), a function conserved from worms to humans. The insect nAChR is a pentamer of α subunits, or a heteromer of α and β subunits and 10 subunits have been reported in Drosophila. Vast diversity is generated through different subunit assembly, RNA editing and alternative splicing. Thousands of subtle and noticeable pharmacological and electrophysiologically diverse receptors could be assembled. In insects, nAChR’s are targets of insecticides used to control pests. Chapter two describes work on the characterization of nAChR subunit genes in the central nervous system (CNS) of an embryo and larval D. melanogaster stages through in-situ hybridization, Fluorescent in-situ hybridization (FISH) and enhancer studies. Expression of 7 of the nAChR subunits (Dα1, Dα2, Dα3, Dα5, Dα6, Dα7 and Dβ2) was observed in CNS of embryo and larval CNS tissues. Beside the CNS, expression was also observed in other tissues, such as the ring glands (Dα1, Dα5, Dα7 and Dβ2) suggesting a role for these in the developmental biology of Drosophila. Salivary gland expression was observed for Dα7 subunit while larval fat body and adult hemolymph expression was observed for the Dβ3 subunit gene suggesting novel roles for these nAChR subunits. Building on the expression of these individual nAChR subunits, co-localization was also observed for Dα1/Dα2 and Dα1/Dβ2 subunit genes in larval CNS using FISH. In the third chapter a new approach was taken using RNAi as a tool for predicting insecticide resistance before it happens and finding new insecticide targets. Ten of the nAChR subunit genes were knocked-down using RNAi lines in the CNS of Drosophila. Results suggest that Dα6 is the only subunit targeted by the insecticide spinosad. Also individual knockdown of the Dα1 and Dβ3 subunits show significant sensitivity to spinosad, suggesting some form of compensation mechanism for these nAChR subunits. Conclusions from this work were that RNAi is an excellent tool in Drosophila (due to the availability of RNAi lines) in predicting resistance to insecticides, and prior testing of compounds could assist with better management of resistance development in insect pest species as insecticide targets are commonly conserved among insect species. The fourth chapter examines a negative cross-resistance of spinosad and nitenpyram resistant strains and by using mixtures of these insecticides to detect possible synergistic interactions. Negative cross-resistance was confirmed in earlier studies carried out by T. Perry (2005); a nitenpyram resistant mutant Dα1ems1 was observed to be sensitive to spinosad and a spinosad resistant mutant Dα6ems6 showed sensitivity to nitenpyram insecticide. My work using a number of mixture ratios found significant synergism between nitenpyram and spinosad insecticides at a ratio of 75 to 1. This synergistic ratio was found to be effective against the target site resistant mutants of nitenpyram and spinosad and also against a metabolic resistance mechanism to nitenpyram, indicating that mixtures can overcome both metabolic and target site resistances. My discussion chapter (Chapter 5) evaluates the expression studies and possible functions associated with the expression patterns observed in a particular tissues/life stage of Drosophila. It examines the advantages of some of our techniques such as RNAi as a fast method of predicting resistance. The implications of the negative cross-resistance relationship between spinosad and nitenpyram insecticides and the use of these two in mixtures are discussed with reference to resistance management and touches on future directions and ideas of practical implications of this in the field.
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    Cytochrome P450 gene expression in Drosophila melanogaster
    Chung, Hock Wee Henry ( 2008)
    Present in almost all living organisms, cytochrome P450s form one of the biggest enzyme superfamilies. They are versatile biocatalysts, capable of performing a range of biochemical reactions and are involved in a wide spectrum of biological functions. The vinegar fly, Drosophila melanogaster, has 85 P450s in its sequenced genome. Six of these have been found to catalyse the synthesis of the important insect molting hormone, 20-hydroxyecdysone and a handful have been implicated in insecticide resistance. The other P450s remained largely uncharacterised. In the first half of this thesis, the expression patterns of P450s in the D. melanogaster genome were characterised by in situ hybridisation at the third instar larval stage. Most P450s have defined expression patterns at this stage of development. A majority of P450s are expressed in the midgut, Malpighian tubules and fat body, tissues that are involved in the metabolism of xenobiotics. Other P450s are expressed in specific tissues, such as the prothoracic glands, the salivary glands and the gonads, where they might have roles in development or reproduction. In particular, Cyp6g2 is expressed in the corpus allatum (CA), where it could play a role in juvenile hormone synthesis. An RNAi lethality screen using lines that were available from the Vienna Drosophila RNAi Centre identified a number of P450s which are essential for development and viability. In the second half of the thesis, the transcriptional regulation of a P450 involved in insecticide resistance, Cyp6g1, was investigated. Cyp6g1 was regulated by two discrete cis-regulatory modules/enhancers, one controlling expression in the Malpighian tubules and one controlling expression in the midgut and fat body. Phenobarbital induction of Cyp6g1 is tissue-specific and is mediated by a fragment in the 5’ regulatory region that interacts with both enhancers. Characterisation of the long terminal repeat (LTR) of the Accord transposable element in the 5’ region of Cyp6g1, present in insecticide resistant populations, shows that the Accord LTR contains cis-regulatory elements which increase expression of Cyp6g1 in the fat body, midgut and Malpighian tubules, and contribute to insecticide resistance in these populations. This study shows that the diverse tissue distribution of different P450s in D. melanogaster is related to the diverse biological functions of the enzymes encoded. This is exemplified by the detailed examination of the regulation of the insecticide resistance-conferring P450, Cyp6g1. Its expression pattern reflects its detoxification function in the fly. The role of transposable element insertions in changing gene expression patterns and contributing to selectable variation in genomes is also demonstrated through the Cyp6g1 study.