Genetics - Theses
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ItemThe genetic basis of resistance to the Ryanodine Receptor modulator chlorantraniliprole in Drosophila melanogaster( 2017)The development of synthetic insecticides in the mid 20th century lead to a revolution in pest control. However, issues with environmental toxicity, adverse human exposure and insecticide resistance have meant new safer alternative pest control methods are required. Chlorantraniliprole belongs to a promising new class of insecticides that exert control by targeting the Ryanodine Receptor. As this class, the group 28 synthetic diamides, has a unique chemistry and a mode of action that is distinct from most insecticides, its market share has rapidly increased since it was first introduced in 2007. Here, I use genomic, transcriptomic and phenotypic analyses of the Drosophila Genetic Reference Panel (DGRP) to examine the way chlorantraniliprole interacts with an insect’s biology. This research reveals that a novel muscle- associated gene, Stretchin Myosin Light Kinase, is strongly associated with resistance in the DGRP. In addition, a co-expressed set of detoxification enzymes, under control of the Cap ‘n Collar/Keap1 pathway were found to be constitutively up-regulated in a subset of the DGRP and that their transcriptional abundance was correlated with survivorship on chlorantraniliprole. Transgenic ‘knock up’ of one of these putative detoxification enzymes, Cyp12d1, confers increased resistance to chlorantraniliprole and resistance to the related compound cyantraniliprole. Furthermore, a lab selection experiment based on a large Australian population of D. melanogaster also confirms an association between Cyp12d1 and resistance. This contributes to a growing body of evidence suggesting that cytochrome P450 enzymes play a role in chlorantraniliprole resistance. Through the quantitative genetic approaches employed, it is possible to demonstrate that the genetic architecture underpinning resistance changes with dose. Furthermore, as the DGRP was established before the introduction of chlorantraniliprole this study demonstrates that alleles of large effect are pre- existing in naïve populations and such alleles may increase in frequency as this class of insecticides become more widespread. Finally, this study illustrates the systems genetic approach offers unprecedented power to understand the biology perturbed by insecticides.
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ItemThe genetics of resistance to lufenuron in Drosophila melanogaster( 2005-02)The rise of large scale agriculture in the 20th century created the need for effective strategies to control insect pests. Treatment with chemical insecticides has been a weapon of choice, but the inevitable evolution of resistance has followed in many insect species. Resistance represents a major challenge, not only for agricultural production, but also for environmental preservation and human health. Two major options for resistance have been identified, and these are target-site based and metabolic-based resistance. Much insecticide resistance research focuses on identifying these mechanisms through genetic and molecular analysis. The insecticide lufenuron is the focus of this study. It belongs to a novel insecticidal group called the insect growth regulators, which were introduced in 1970s as highly selective insecticides with low vertebrate toxicity. Resistance to lufenuron in the non-pest species Drosophila melanogaster has been observed in field populations, despite the lack of field usage of lufenuron (Wilson & Cain, 1997; O’Keefe, 1997). This study has taken advantage of this phenomenon to investigate resistance mechanisms in natural populations. At least two detoxification mechanisms were identified. (For complete abstract open document)
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ItemNitenpyram resistance in Drosophila melanogaster( 2012)The neonicotinoid insecticides are effective in controlling a variety of insect pests in the field. They target the nicotinic acetylcholine receptor, a cation channel in the central nervous system. After years of usage, high levels of resistance to these insecticides are beginning to emerge. This requires greater understanding of the activity of these insecticides and the function of these channels. The bulk of this study made use of the model organism, Drosophila melanogaster, and the well-defined family of ten nicotinic acetylcholine receptor subunits encoded by its genome. Mutations and null alleles of insect nicotinic acetylcholine receptors have been described but, thus far, all of these are homozygous viable. This is surprising given the high level of conservation of these genes. Part of this study examines viability with loss of function of individual subunits through the use of stably integrated RNAi constructs. Ubiquitous knockdown of any one gene had a significant effect on mortality prior to the adult stage. Knockdown of Dα1, Dα5 or Dβ2 produced the most severe increases in mortality. However, genomic deletions of either Dα1 or Dβ2 were seen to be viable. Deletion of Dα5 resulted in a completely penetrant larval lethality phenotype with all the hallmarks of a possible role in hormonal control of development. Further studies of knockdown flies highlighted possible roles in mating for Dα1, Dα2, Dα6 and Dβ1. RNAi knockdown of individual receptors was also used to investigate the possible involvement of each subunit in mortality induced by the neonicotinoids. RNAi knockdown larvae were exposed to nitenpyram and survival to adulthood scored. Resistance was seen for knockdown of known nitenpyram targets Dα1 and Dβ2, confirming this approach provides enough sensitivity to reveal nitenpyram targets. Resistance was also seen upon knockdown of either Dα3 or Dβ1. In addition to this, knockdown of either Dα6 or Dβ3 produced hyper-sensitivity to nitenpyram. Results from this study are juxtaposed to published associations of nicotinic acetylcholine receptor subunit resistance alleles to propose likely components of the neonicotinoid binding sites. The neonicotinoid insecticides are known to bind at the acetylcholine binding site, which occurs at a cleft formed between two subunits in the mature receptor. The information gained from published work and the RNAi experiments using nitenpyram suggested two likely interfaces that could make up the insecticide binding site. Homology modeling based on the structure of the Aplysia californica acetylcholine binding protein was used to generate three dimensional structures of the two likely interfaces. Binding of several neonicotinoids was simulated and produced a similar binding orientation in a Dα1/Dβ2 interface. Comparison of imidacloprid binding to acetylcholine binding and imidacloprid resistance mutations strongly supports the model presented here. The final part of this study is the genetic mapping of a novel resistance mechanism to nitenpyram isolated in a field collected strain. A previously identified strain of D. melanogaster collected at Cape Tribulation, Australia, had been shown to carry a resistance factor on the third chromosome. P-element mapping and screening of molecular markers identified a resistance locus on chromosome 3L between cytological bands 68c4 and 68d2 that contains 50 genes. The known functions and expression patterns of these genes are considered and possible resistance mechanisms are discussed.
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ItemNicotinic acetylcholine receptors; an examination of expression and insecticide interactions in Drosophila melanogaster( 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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ItemThe role of cytochrome P450s in pyrethroid resistance in the AN02 strain of Helicoverpa armigera( 2003)Helicoverpa armigera is a lepidopteran moth species that is one of the largest agricultural pests in the world. In Australia, it is especially damaging to the cotton industry. Previous research on pyrethroid resistance in Helicoverpa armigera has suggested several resistance mechanisms present in the field. Research on a specific strain of H. armigera, AN02, suggested that resistance to pyrethroids in this particular strain is due to metabolism of the insecticide by cytochrome P450. This thesis describes work undertaken to gain further knowledge of the resistance mechanism present in this strain. Chapter 2 describes expression testing and elimination of the CYP6B cluster of P450s as candidates for resistance in the AN02, which were the most likely candidates available at the time. Chapter 3 describes construction, probing and sequence analysis of a midgut cDNA library of H.armigera to clone more P450 sequences and analyse the general transcriptome of one of the tissues where detoxification is likely to occur. Chapter 4 describes expression testing of new P450 sequences obtained from different sources to determine whether any of them have increased expression that is associated with the resistance genotype. Several new P450 genes were found to have increased expression and are candidates for further study.
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ItemCytochrome P450 gene expression in Drosophila melanogaster( 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.