Genetics - Theses
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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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ItemUsing insecticides to probe nicotinic acetylcholine receptors in Drosophila melanogaster( 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.