Genetics - Theses

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    Roles of nicotinic acetylcholine receptors in development, viability, and insecticide response, in Drosophila melanogaster
    Christesen, Danielle Maree ( 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.