Genetics - Theses
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ItemNovel roles of cytochrome P450 genes in insect development(University of Melbourne, 2009)Cytochrome P450s form one of the largest enzyme superfamilies. They are involved in the catalysis of a number of different chemical reactions and are involved in a wide range of biological processes. In the vinegar fly, Drosophila melanogaster, 85 P450s have been identified. This thesis uses genetic approaches to investigate the roles of specific P450s in different aspects of D. melanogaster development and reproduction. The expression patterns of all 85 P450s in the D. melanogaster y ; cn, bw; sp strain were analysed by RT-PCR and in situ hybridisation during embryogenesis. In situ hybridisation patterns were obtained from 50 out of 65 P450s expressed during embryogenesis. Many novel expression patterns were identified; Cyp28c1 was detected in the salivary glands, Cyp49a1 was detected in the hindgut and Cyp18a1 was detected in the epidermis. These results were compared with larval in situ hybridisation patterns of P450s previously characterised, to identify P450s with conserved expression throughout the life-stage. An RNAi screen was also performed. Of the 60 P450s targeted by RNAi, nine were shown to be lethal (Cyp18a1, Cyp28c1, Cyp306a1, Cyp309a1, Cyp311a1, Cyp314a1, Cyp4c3, Cyp4d2, Cyp4g1, Cyp6g2) and three showed reduced survival (Cyp318a1, Cyp4ac2, Cyp4s3). The function of Cyp301a1 was also investigated using in situ hybridisation and RNAi. Cyp301a1 was detected in the hindgut and epidermis in D. melanogaster y; cn, bw; sp embryonic and adult stages. RNAi knockdown of Cyp301a1 as well as analysis of the Cyp301a1f02301 mutant flies showed disrupted cuticle phenotypes, suggesting that Cyp301a1 has a role in cuticle formation. Cyp6g2 was the only P450 detected in the corpus allatum, the site of juvenile hormone synthesis. RNAi knockdown of Cyp6g2 is lethal at pupal stages and lethality can be rescued by over-expression of D. willistoni Cyp6g2. Cyp6g2 appears to be unique to the Drosophila genus and is highly conserved in all twelve sequenced Drosophila genomes. Cyp6g2 expression fluctuates dynamically during larval and pupal development and is roughly correlated with high JH titres. Knockdown of Cyp6g2 disrupts the expression of other genes known to be regulated by JH, suggesting that Cyp6g2 may have an essential role in juvenile hormone regulation. Interestingly, Cyp6g2 is also expressed in the adult male ejaculatory bulb and was differentially expressed during mating, indicating an additional reproductive role. The evolution of the Cyp307a genes in Drosophila was investigated. The Cyp307a genes are involved the synthesis of 20-hydroxyecdysone, an essential insect moulting hormone. In D. melanogaster, there are two Cyp307a genes; Cyp307a1, which was detected in the yolk cells of the early embryo and in the follicle cells of the female ovary and Cyp307a2, which was detected in the prothoracic cells of the ring gland. Phylogenetic and microsynteny analyses in Drosophila showed a complicated evolutionary scenario for the Cyp307a genes, invoking multiple gene duplications (and subsequent losses) in different lineages. The varied temporal and spatial expression of the Cyp307a genes in Drosophila suggested that they have undergone independent evolution and sub-functionalisation within the Drosophila genus. Finally, this thesis investigated roles of cytochrome P450s in reproduction. Using in situ hybridisation, several P450s were detected in the adult female reproductive tissues. Cyp6a19 and Cyp6a22 were highly expressed in the nurse cells whereas Cyp18a1 was detected in the follicle cells of the developing oocyte. Cyp305a1 was detected in the follicle cells covering the late stage oocyte and in the dorsal region specifying the appendage. Ubiquitous knockdown of Cyp305a1 by RNAi was lethal, suggesting it plays an essential role during development. RNAi of Cyp305a1 in the follicle cells, showed defects in nurse cell dumping and dorsal appendage formation. Several P450s were also detected in the adult male reproductive system, with Cyp312a1 expressed highly in the developing spermatocytes of the male testes. RNAi knockdown of Cyp312a1 was viable and Cyp312a1 RNAi progeny showed normal testes development. However Cyp312a1 RNAi males were less fertile, which may be due to defects in sperm maturation or protection in the female reproductive tract.
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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.
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ItemExpression and function of five ligand-gated chloride channel genes of Drosophila melanogaster( 2012)Annotation of the genome of the vinegar fly, Drosophila melanogaster, has identified 12 ligand-gated chloride ion channel (LCCH) subunit genes. The ligands for the receptors produced from these LCCH subunit genes are the neurotransmitters GABA (γ amino butyric acid), glutamate, histamine and glycine. The ligands of some subunits are yet to be discovered. The GABA-gated LCCH subunits have sequence homology with vertebrate GABAA receptors that have vital functions in nervous system development and neurotransmission. The Rdl gene of D. melanogaster encodes a LCCH subunit. This gene came to prominence when it was found that a naturally occurring allele, in D. melanogaster and many insect pest species, conferred high level target site resistance to the insecticide dieldrin. Subsequently the Rdl (Resistance to DieLdrin) gene and its product (RDL) have been intensively investigated. The Rdl gene has been associated with sleep, learning and memory, epileptic seizure and phenotypes due to defects in Drosophila Fragile X mental retardation gene function. There are four other D. melanogaster genes that been reported to encode subunits that assemble into GABA-gated cation channels (Lcch3 and Grd) or have the GABA-binding consensus sequences found in vertebrate GABA receptor subunits (CG8916 and CG12344). These other genes are also likely to play important functional roles in neurotransmission and/or development but, to date, there has been a striking lack of research focused on elucidating their functions. In this thesis two approaches are combined to examine the function of these genes. In the first of these, the expression of the Rdl, Lcch3, Grd, CG8916 and CG12344 genes is examined using in situ hybridization. Further, the cloned endogenous promoters for the five genes are used to drive the expression of the green fluorescent protein (GFP) using the GAL4/UAS system (Chapter 2). The spatial and temporal expression patterns for these genes provide some clues to their function. The second approach uses RNA interference to knockdown gene expression to generate mutant phenotypes indicative of gene function (Chapter 3). A number of phenotypes are described here – defects in larval forward locomotion and wing inflation, necrotic tissues and lethality. The links between these phenotypes and the function of the five genes are carefully considered. While it is not the major focus of this thesis, the applications of the research described are considered. The RDL protein has been targeted with a range of insecticides. This thesis identifies two other GABA-gated cation channels (GRD and LCCH3) as potential insecticide targets. Given the similarities between the five GABA-gated LCCH subunits and their human counterparts, some fruitful lines of research that may positively impact the further study of human biology and health are identified. Most importantly, the research reported here provides a foundation of knowledge and reagents that will be of great value in an ongoing systematic analysis of the role that these five GABA-gated LCCH subunits play in insect development and neurotransmission.
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ItemEvolution of Wolbachia-Drosophila interactions and implications for Wolbachia-based biocontrol( 2010)Wolbachia is a maternally inherited α-proteobacteria that lives an obligate intracellular lifestyle in its eukaryotic hosts. Wolbachia pipientis was first identified in the 1920’s in the mosquito Culex pipiens; Wolbachia has since been detected in a broad range of hosts. Wolbachia induces a number of diverse reproductive manipulations in many of its hosts, implicating Wolbachia in control strategies for target species. However, changes in host bacterial interactions can evolve toward mutualism, and this requires investigation in target systems. This first part of this work examines changes in the Drosophila simulans-Wolbachia Riverside system in California. This system, studied for a period of more than two decades, provides a rare example in nature of a symbiotic relationship shifting from parasitism to mutualism. In my study, no changes in overall estimates of infection frequency, maternal transmission or cytoplasmic incompatibility were found, however there appears to be heterogeneity in maternal transmission rates and the ability of males to induce cytoplasmic incompatibility (CI). The source of CI variation in one isofemale line was subsequently investigated through reciprocal backcrossing with an old Wolbachia-infected strain, Riv88, and the variation was attributed to the Wolbachia genome. Further investigations are required to determine the extent of CI variation in the wild. These findings significant implications for the many applied projects that intend to exploit Wolbachia for its ability to manipulate host reproductive systems. The second part of this work investigates the extent to which evolution of a host-bacterial symbiosis can influence the effectiveness of control strategies using Wolbachia. A particular strain of Wolbachia, named popcorn (wMelPop), has been isolated for its ability to reduce host longevity. This bacterium has the capacity to alter the age structure of the target host populations. As many vector-borne diseases (eg: Dengue Fever, Malaria) require an incubation period within the vector prior to transmission to the final host, a reduction in the lifespan of vector populations has the potential to lower transmission levels. The long term stability of this bacterial strain has been investigated in its native host (Drosophila melanogaster), and a novel host (D. simulans). The longevity-shortening phenotype and several life-history traits that might influence the effectiveness of control strategies have been examined. Host genetic background and the indirect selection on the longevity phenotype (via early or late fecundity selection) were found to influence multiple life-history traits in these species. The source of this variation in D. melanogaster is attributed primarily to the host, but can also be influenced by Wolbachia. Strong host background effects were noted in D. simulans, which highlights the importance of testing multiple backgrounds for suitability of use in applied projects. This work has allowed for greater understanding of the potential for evolution within host-symbiont systems, and can be used to provide a framework for undertaking projects that will use Wolbachia as biological control agents. Future projects should consider the potential for evolution in detail under laboratory conditions before control strategies are implemented in the wild.