Genetics - Theses
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ItemNo Preview AvailableRoles of nicotinic acetylcholine receptors in development, viability, and insecticide response, in Drosophila melanogaster( 2021)The majority of excitatory neurotransmission in the insect brain occurs via nicotinic acetylcholine receptors (nAChRs), however knowledge of which nAChR subunits may be required in specific neurons is virtually absent. The ubiquity of nAChRs in the insect brain also makes them ideal molecular targets for many neuroactive insecticides. Mutations in specific nAChR subunits can confer high levels of resistance, but the fitness costs that may be associated with resistance alleles or with low-dose insecticide exposure are not fully understood. This thesis examines two Drosophila nAChR subunits with the most severe loss of function phenotypes, with a focus on their endogenous functions and their roles in insecticide response. Chapter Two investigates the role of the Da5 subunit in larval development. Preliminary observations had indicated that loss of Da5 causes larval mortality and is associated with precocious wandering and moulting phenotypes. Here, these phenotypes are quantified and found to be associated with loss of ecdysis triggering hormone. Cell types requiring Da5 were also narrowed-down to potentially include the prothoracic gland cells, or the neurons innervating the prothoracic gland. In insecticide exposure assays, loss of Da5 was shown to not confer altered response to spinosad, suggesting that Da5 may not be contributing to the primary spinosad target. In Chapter Three, loss of the Db1 subunit is shown to result in pleiotropic consequences, including severely shortened longevity, reduced male courtship, limited locomotion, and unsuccessful wing expansion. Since wing expansion is controlled by a well-characterised hormone (bursicon) in a small and well-defined subset of neurons (the CCAP neurons), this phenotype was examined further. Removal of Db1 specifically from CCAP neurons using somatic CRISPR was sufficient to disrupt wing expansion and loss of Db1 was shown to cause loss of the hormone bursicon. Together, these experiments identify CCAP neurons as a specific subset requiring Db1 for normal function. Chapter Four extends findings from the previous chapter, by testing whether alternative Db1 alleles, and a non-Drosophila b1 subunit orthologue, can rescue the Db1 loss of function phenotypes. Db1 cDNA rescue constructs containing the amino acid replacements R81T (found in resistant populations of aphids), and R81Q (naturally occurring in insensitive arachnids), were both found to rescue all elements of the Db1 phenotype, but only R81Q was found to confer high levels of imidacloprid resistance. The aphid subunit Mpb1 was also able to rescue loss of Db1, and fully restored sensitivity to imidacloprid, revealing substantial functional conservation between the b1 subunits in these two species. This work provides a platform for studying resistance-conferring amino acid replacements in pest nAChR subunits within the Drosophila model. Understanding the endogenous roles of nAChR subunits will be essential for characterising the function of every pathway in the insect brain. By characterising the roles of Da5 and Db1, this thesis provides great insight into the fitness costs insects may endure when evolving insecticide resistance. It also reveals the developmental and behavioural pathways that may be affected when pest and non-pest species are chronically exposed to the low doses of insecticides that contaminate the environment.
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ItemIn vivo functional characterization of nicotinic acetylcholine receptors in Drosophila melanogaster( 2018)Nicotinic acetylcholine receptors (nAChRs) are responsible for fast excitatory synaptic transmission in insect central nervous system. Their role as targets for commercial insecticides have resulted in extensive studies on their structure and pharmacological properties. However, many other aspects of their fundamental biology remain less understood. For example, what behaviours are underpinned by the activity of nicotinic acetylcholine receptors? Here, we used reverse genetics to address this question. The precise genome editing power of CRISPR/Cas9 technology was used to generate a collection of Drosophila melanogaster lines harbouring precise genomic deletions of the genes of interest, including the subunits for the nicotinic acetylcholine receptors as well as a couple of their accessory proteins. The overall strategy was to remove as much as of the genomic locus as possible by having two sgRNAs directing Cas9 to cut at the 5’ and 3’ ends of the gene’s coding sequence and relying on non-homologous end joining repair to ligate the termini together creating a deletion. In total, nine knockout strains were generated for four genes, successfully removing genomic sequences ranging from 4 to 83kb in length. For three genes, Dα4, Dα6 and DmRIC3, the same allele was recapitulated for three backgrounds. The role of nAChRs in regulating sleep behaviour in vinegar flies was investigated using null alleles of the receptor subunits. For seven of the ten subunits, flies harbouring null alleles were viable as adults for behavioural assays. All mutants showed changes in total sleep amount compared to their controls, which most strongly correlated with changes in sleep episode duration. Additionally, genotypes carrying partial deletions or point mutations displayed different sleep changes, suggesting that allelic variation within subunits can yield different phenotypes. These data confirmed a role in sleep regulation for most nAChR subunits. Furthermore, the role of the nAchR accessory proteins were considered. Lines with a deletion of the nAChR-specific chaperone DmRIC3 responded to two commercial insecticides in similar manner to loss of the subunit Dα1. Those lines also phenocopied sleep behaviour of flies lacking receptor subunits. This is the first in vivo evidence of the functional significance of DmRIC3 to nAChRs in D. melanogaster. Altogether, these results show that significant behavioural changes might be considerable fitness costs beyond viability for resistant alleles of genes with important functions in the central nervous system such as nAChRs. However, resistance could still arise from disruption to other proteins interacting and regulating nAChRs with less severe costs.
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ItemProbing insecticide biology using Drosophila melanogaster( 2017)Insecticides are often used to control insect pests, but resistance to these chemicals arises quickly, leading to agricultural losses and public health concerns. Understanding how insects cope with insecticides is necessary when designing rational pest management strategies, but much still remains unknown regarding the fate of insecticides once inside the body. Furthermore, the genetic variation that governs an insects ability to survive insecticide exposures has not been fully described. Here, a 3 pronged approach is applied to study insecticide biology using the model insect Drosophila melanogaster. First, an acute, sub-lethal insecticide response assay was developed, which provided information complementary to that obtained from more common toxicology assays. In particular, behavioural response observed in a hyper-resistant target site mutant suggests additional target sites for the insecticide spinosad. This bioassay was then applied in a forward genetics approach to describe the genetic basis of resistance to the insecticide imidacloprid. This approach identified a variety of neuronal genes and the previously identified drug metabolizing enzyme Cyp6g1, which was explored through genetic manipulation. Finally, a reverse genetics approach was employed in order to study the effect of an ABC transporter protein Mdr65 on insecticide resistance. Removing the gene made the insects more susceptible to a subset of the insecticides tested, and this was confirmed with genetic and chemical complementation tests. These data provide information both on the genetics and kinetics of insecticide biology. Such information will help to better understand insecticide resistance and design rational resistance management strategies.
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ItemPseudogenes and neutral evolution in Drosophila melanogaster( 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.