Genetics - Theses

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    Using insecticides to probe nicotinic acetylcholine receptors in Drosophila melanogaster
    SOMERS, JASON ( 2015)
    Insecticides remain the most effective means of insect control for both our personal protection and for the protection of our food and economic crops. The knowledge gained through the study of current and past insecticides can be a valuable tool in both the design of new, more effective insecticides and as a guide for integrated pest management. To best utilise a new insecticide and for it to retain field efficacy, its mode of action on its molecular target must be thoroughly understood. For this, a genetically tractable model organism can be used to enhance the understanding of insecticide:insect interactions through characterisation of resistance alleles, analysis of the insecticide target(s) and other resistance mechanisms. The advancement of genome engineering technology and the increasing availability of pest genome sequences has increased the predictive and diagnostic capacity of the Drosophila model. The Drosophila model can be extended to investigate the basic biology of the interaction between insecticides and the proteins they target. In this study, the vinegar fly, Drosophila melanogaster, has been used to identify and manipulate insecticide resistance genes. Recently an in vivo system was developed that permits the expression and study of key insecticide targets, the nicotinic acetylcholine receptors (nAChRs), in controlled genetic backgrounds. Rescue of the spinosad resistance phenotype in the Dα6 loss of function mutant, was possible with not only individual isoforms of this gene, but also with pest α6-like orthologues. It has also been found that a chimera of the Dα7 N-terminal and Dα6 C-terminal region is able to rescue the response to the insecticide spinosad. In this study, an incompletely dominant, spinosad resistance mechanism that may evolve in pest species is examined. First generated using chemical mutagenesis, the Dα6P146S mutation was recreated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas9 system, the first reported use of this technology to introduce a resistant mutation into a controlled genetic background. Both alleles present with the same incompletely dominant, spinosad resistance phenotype, proving the P146S replacement to be the causal mutation. The proximity of the P146S mutation to the conserved Cys-loop indicates that it may impair receptor gating. The Dα6 in vivo expression system was used here to assist in characterizing this dominant allele. The results of this study enhance our understanding of nAChR structure:function relationships, in particularly the interaction between spinosad and the Dα6 subunit. A complete in vivo rescue model was developed here for analysis of the Da1 subunit. A Dα1 deletion was generated using ends-out gene knockout technology and this knockout was found to be highly resistant to neonicotinoid insecticides. By combining this deletion with the GAL4:UAS binary expression system this study was able to rescue the phenotype of susceptibility to neonicotinoids as well as confirm the resistance potential of two nAChR subunits from the pest species, Helicoverpa armigera. This system is also used to investigate other phenotypes of the Dα1 deletion. The endogenous role of the nAChRs may enhance our understanding of why these are such effective insecticide targets, but also why they appear to be functionally redundant in terms of insect viability. Knowledge of other phenotypes present in lossof function mutants gives an insight into their function as well as hinting at fitness costs that may be associated with their loss. Targets with important roles are less likely to evolve resistance mutations if they also impact endogenous functions. If the target does have an important function, resistance modifications may lead to decreased fitness that would not be able to persist in natural populations without insecticide selection. A phenotype for the Da1 subunit was discovered whereby it appears to play a role in Drosophila courtship and mating behaviour. Although this allele can be cultivated in laboratory conditions, it would cause dramatic fitness effects under field conditions. Field evolved resistance mechanisms have been identified for both insecticides classes used in this study. Furthermore the neonicotinoid, imidacloprid, has recently been implicated in colony collapse disorder that is currently impacting honeybee populations. The results in this study provide information about the basic biology of the molecular targets of these insecticides. This information may help extend the life of these insecticides through more efficient use or perhaps provide ideas for new, more specific insecticides.
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    Pseudogenes and neutral evolution in Drosophila melanogaster
    Bardsley, Lisa M. J. ( 2013)
    An understanding of the rates and patterns of neutral evolution is important for estimating divergence times and for recognising selection acting upon the genome. In order to characterise neutral evolution it is necessary to identify sequences that are evolving free from selective constraint. This has proven difficult in Drosophila, where sites such as synonymous sites, introns, and untranslated regions of genes have been shown to have selection acting upon them. Pseudogenes are inactive copies of genes that are by definition functionless, and are thus ideal candidates for the study of neutral evolution. Historically few pseudogenes have been known in Drosophila, and many sequences that have been thought to be pseudogenes have been found to be functional. The advent of the genomics era has allowed for the identification of many more potential pseudogenes. In this thesis I study these in order to identify likely genuine pseudogenes, and use these to characterise neutral evolution in Drosophila. The first step of this project was to identify a list of genuine pseudogenes. Two pseudogene datasets were used: those identified in a paper by Harrison et al., (2003), and those listed on the Drosophila genome database Flybase. A number of techniques were used to study these pseudogenes, including cDNA analysis, conservation analysis, and resequencing. The results of this study showed that many of these sequences were not genuine pseudogenes and had been incorrectly assigned due to incorrect genome annotation, unknown splicing patterns, and polymorphic inactivating mutations. In total 73 likely Drosophila melanogaster pseudogenes were identified. The technique Gene Identification by Nonsense-Mediated Decay (GINI) (Noensie & Dietz, 2001) was investigated as a possible technique for identifying new pseudogenes. This involved feeding Drosophila larvae drugs known to inhibit nonsense-mediated decay and then determining whether this resulted in an upregulation of transcripts known to contain premature termination codons (PTCs). Caffeine was found to result in the upregulation of 4 of 6 PTC-containing transcripts, most notably those with longer 3’ UTRs. This technique thus seems promising for the identification of new pseudogenes. Following this, 47 pseudogenes were resequenced in three populations. It was found that ancient pseudogenes had higher levels of nucleotide diversity than recently inactivated genes and new pseudogenes, presumably due to their time since inactivation and pseudogene population size during this time. Various aspects of pseudogene evolution were characterised including nucleotide diversity, mutation patterns, and FST, allowing us to gain a better understanding of population structure and the background mutational patterns of the genome. Finally, I investigated several hypotheses as to why the Esterase-7 gene might have a large proportion of inactivated alleles in natural populations of D. melanogaster. I concluded that the selective constraint acting on Esterase-7 has likely been relaxed in D. melanogaster relative to other species, and Esterase-7 may be in the process of becoming a pseudogene. This gives us key insights into the pseudogenisation of functional genes by mutation and genetic drift - mutations gradually occur and spread throughout the population while functional copies still remain in the population.
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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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    Dissection of the genetic and physiological basis of desiccation resistance in Drosophila melanogaster using laboratory selection
    de Garis, Sarah Elizabeth ( 2011)
    Desiccation stress is a major factor underlying the distribution, abundance and evolution of insect species. This study utilises laboratory selection to dissect the physiological and genetic basis of desiccation resistance in a population of D. melanogaster recently collected from the field. The first part of this thesis assays selected and control flies for a series of physiological traits. Selected flies were found to lose water at a slower rate than their controls when exposed to desiccation stress, in keeping with previous investigations. Another major physiological mechanism underlying the selection response was increased water storage, possibly mediated via enlarged glycogen reserves. This study is the first to demonstrate that these traits can evolve in response to desiccation selection in fly lines recently derived from nature. Dehydration tolerance and whole-body lipid content however did not contribute towards the selection response, a finding consistent with most prior studies. The second part of this study investigates whether desiccation selection produced correlated responses in other environmental stress traits. In keeping with previous experiments, a strong correlated response was observed between desiccation and starvation resistance, suggesting that the two traits are shaped by at least partially overlapping physiological mechanisms. In contrast this investigation found no correlated response between desiccation resistance and cold tolerance, as measured by both cold mortality and chill coma recovery. The data surrounding heat tolerance were more complex; while higher heat knockdown resistance failed to evolve in response to desiccation selection, increased heat mortality and, to a lesser extent, Critical Thermal Maximum (CTMAX) did. This investigation also aimed to assess whether flies selected for increased desiccation resistance in the laboratory showed an increase in an aspect of field fitness under dry conditions. Female selected flies were better able than their controls to reach food resources following release into a natural environment but this was not the case for males. This result indicates that though laboratory selection for desiccation resistance can result in ecologically relevant evolutionary change, levels of field fitness are not always accurately predicted by laboratory-based assays. Finally, tiling microarrays were used to detect sequence variation between selected and control populations. Six hundred and seventy one Single Feature Polymorphisms (SFPs), located across all chromosomes, were found between the two treatments, none of which correspond to known desiccation resistance candidates. Sequence divergence was validated via sequencing for eight genes: CG7084, beat-VII, CG7638, omega, Dystrophin, SNF4Agamma, mol and CG14304. These genes may become solid desiccation resistance candidate genes if they are found to be associated with the trait in independent, natural populations.
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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.