1,721,105 research outputs found
Genetic control of the diamondback moth (Plutella xylostella L.)
Full text linkInsect pests represent major threats to food production, biodiversity conservation, and human and animal health. Currently, the most widespread strategy to control their populations is through the spraying of synthetic chemical insecticides. However, the overuse of these compounds has had significant negative environmental consequences. Additionally, our reliance on insecticides has resulted in major reductions in their efficacy through pest-evolved resistance. To successfully manage insect pests, while avoiding environmental degradation, thus requires the development of novel, more sustainable, pest management strategies. Recent advances in our understanding of recombinant DNA methods and molecular biology have allowed the application of transgenic tools to pest management. Here, synthetic genes can be engineered, transformed into the genomes of pest species, and transported into wild target populations through the natural mating behaviour of the insect. A strategy in which these transgenes are lethal to those insects inheriting them in the field is known as RIDL – Release of Insects carrying a Dominant Lethal. A variant of RIDL limits this lethality to females – female specific RIDL (fsRIDL) – which explicitly targets the reproductive capacity of a target population. The aim of this thesis is to investigate the application of such an fsRIDL strategy to the diamondback moth (Plutella xylostella L.). This economically important pest of brassica crops is highly adept at developing resistance to insecticides and is considered extremely difficult to manage effectively. I present findings which demonstrate the power of diamondback moth lines transformed with fsRIDL transgenes to eliminate target pest populations, and combine synergistically with other transgenic control strategies such as Bt crops in counteracting the evolution of pesticide resistance. Additionally, an exploration into an alternative gene expression system to that used in current RIDL strategies – the Q system – suggests that not all expression systems will be suitable for transgene control within this highly specific framework. It is hoped that this work will contribute towards the effective control of the diamondback moth, and form a model for the sustainable control of other lepidopteran species through genetic pest management
Genome engineering and gene drive in the mosquito aedes aegypti
Full text linkGenetic control strategies are a novel method for reducing populations of pest insects such as the yellow fever mosquito Aedes aegypti, a major vector of several important arboviral diseases. This thesis describes efforts to develop new tools to engineer the Ae. aegypti genome and to better understand existing tools, and furthermore to use these to engineer a gene drive system in Ae. aegypti. The piggyBac transposon was found to be extremely stable in the germline of Ae. aegypti, and transposons engineered into the germline could not be remobilized with either an endogenous or exogenous source of piggyBac transposase. Conversely, somatic remobilization of piggyBac transposons was found to be readily detectable in the presence of a source of active transposase, the first report of such remobilization in Ae. aegypti. Toward new tools for genome engineering, the site-specific integrase from the phage φC31 was successfully used to promote exchange between a transgene cassette inserted into the genome of Ae. aegypti and a cassette in a plasmid vector, in the first demonstration of recombinase mediated cassette exchange technology in a pest insect species. The integrases from phages φRV1 and Bxb1 were not found to be active in the germline of the mosquito. Finally, development of a gene drive system in Ae. aegypti using an RNAi-mediated killer-rescue mechanism was attempted. Tissue-specific expression of tTAV-regulated-toxic effectors genes, using the promoter regions of the blood meal induced genes Carboxypeptidase A-1, 30Kb and Vitellogenin A, was possible, but sex-specificity was not achieved. A blood meal inducible lethal phenotype was not possible using the chosen promoters, with expression of the effectors either leading to death in early development or to a sublethal phenotype. RNAi against tTAV fused to the Mnp fragment of the dengue virus’ genome was tissue specific, but was found to be highly effective in the fat body suggesting that the Vitellogenin A was the best candidate for the engineering of killer-rescue systems in the mosquito
The genetic control of Aedes aegypti
Full text linkIn the last century, we have observed the introduction, establishment and expansion of mosquito-borne diseases into diverse new geographic ranges. The utility of genetically engineered mosquitoes as tools to decrease the burden of disease by controlling disease-transmitting vectors is being evaluated. The work in this thesis contributes to this goal by exploring mechanisms to spread (or 'drive') anti-pathogenic traits (i.e. disease refractoriness) into target populations through the use of an engineered gene drive system in Aedes aegypti, and by developing additional tools for the safe, reliable, and targeted transformation of these mosquitoes for field release using a novel site-specific cassette exchange mechanism. The proposed gene drive system is underdominance-like as it relies on the inheritance of a pair of trans-suppressing lethal constructs, and uses a novel design to help tackle the 'linkage problem', which is the potential dissociation of the drive system and its 'cargo' anti-pathogenic gene(s). One component of this proposed gene drive system is a lethal or fitness-reducing gene. A range of effector proteins with different biochemical modes of action was screened for their suitability in this system. Effectors that looked promising in this initial screen were evaluated further for their phenotypes when expressed under the control of selected blood-meal inducible promoters. One combination gave the interesting and novel phenotype of temporary blood-meal-induced paralysis. Partial suppression of effector expression was achieved by co-expressing a hairpin RNA for RNA interference, however it proved difficult to combine adequate fitness penalty and rescue to the degree required for a field-usable system. The cassette exchange system combines the ΦC31-att integration system, and Cre or FLP-mediated excision to remove extraneous sequences introduced as part of the site-specific integration process. This provides a useful new tool for genome manipulation. Complete cassette exchange was achieved and the absence of any obvious fitness costs or positional effects in two docking strains make these lines good candidates for both research and generation of new transgenic strains for genetic control of Ae. aegypti
Engineered genetic sterility of pest insects
Full text linkIn the light of increasing pesticides resistance in agricultural pests and in insect vectors of human diseases, leading to the rise in occurrence of mosquito-borne diseases, new, efficient and environmentally friendly methods of pest control are needed. Sterile Insect Technique (SIT), relying on mass releases of radiation sterilised males to reduce reproductive potential of target pest populations, although not new, offers an alternative to the use of pesticides and is an environmentally non-polluting method of insect control. Many insect species, however, are not very amenable to classical SIT, due to detrimental side-effects of radiation treatment. We propose a new method, a genetically engineered modification of classical SIT, replacing radiation with genetically induced sterility. Based on conditional expression of male-germline targeted nucleases which introduce double strand breaks into the male germline DNA to render males sterile, this method emulates SIT mechanism, at the same time eliminating radiation and associated detrimental side-effects. Different variants of such a system were investigated in this project, eventually leading to the creation of functional conditional male-sterility systems in two model organisms – the Yellow fever mosquito, Aedes aegypti and the Mediterranean fruit fly, Ceratitis capitata. Both systems utilise chimeric nuclease composed of protamine and FokI cleavage domain fusion. The sperm-specificity and the conditionality of the sterile phenotype have been achieved through the use of tetracycline repressible expression system driven by the β2-tubulin promoter in Ceratitis capitata and by the Topi promoter in Aedes aegypti
Wolbachia-mosquito interactions and engineered female-specific lethality for Aedes-arbovirus control
Full text linkAedes aegypti and Aedes albopictus are the primary vectors of dengue (DENV) and chikungunya (CHIKV) viruses, causing millions of infections annually. Novel genetic mosquito control methods are being trialled, including population replacement using disease refractory mosquitoes transinfected with the intracellular bacterial endosymbiont Wolbachia, and population suppression by release of male mosquitoes carrying self-limiting genes. The underlying cause of Wolbachia-induced DENV refractoriness is unconfirmed. In Chapter 2, I examine in Ae. albopictus the hypothesis that reactive oxygen species (ROS) induced immune activation plays a role, but neither ROS nor innate immune pathways were upregulated. In Chapter 3, an untargeted analysis of the Ae. albopictus lipidome showed it is significantly altered by Wolbachia infection, highlighting sphingolipids as candidates for future investigations into DENV-refractoriness. Genetic constructs for population suppression are better characterised than Wolbachia-host interactions but incompletely described modes of action and reliance on random genome integration to control expression fails to take full advantage of engineering approaches to design optimal genetic control phenotypes. In Chapter 4, I engineer female-specific, late-acting lethality in Ae. aegypti using specific effectors crossed with candidate pupal-stage promoters and a doublesex cassette causing sexspecific splicing. In Chapter 5, I transform and characterise Ae. aegypti lines producing sex-specific Drosophila melanogaster alcohol dehydrogenase (Adh) to evaluate potential for early-stage inducible genetic sexing. No effective lines were produced but I suggest future design alterations. Overall, this thesis contributes to understanding the molecular mechanisms underlying two distinct genetic control strategies in Aedes mosquitoes. It promotes the use of that mechanistic knowledge to engineer optimal traits for control and for improving mass-rearing methods to meet the clear need for effective and economical area-wide control of arbovirus vectors
Genetic control of the olive fruit fly, Bactrocera oleae
Full text linkThe olive fruit fly, Bactrocera oleae, (Rossi) (Diptera:Tephritidae), is a key pest of olive crops. The sterile insect technique (SIT) is an environmentally benign and species-specific method of pest control, aiming to reduce the reproductive potential of a wild population through the mass-release of sterile insects. Previous olive fly SIT trials, involving the release of gamma-ray sterilised mixed-sex populations, achieved limited success. Key problems included altered diurnal mating rhythms of the laboratory-reared insects, leading to assortative mating between released and wild populations, and low competitiveness of the radiation sterilised mass-reared flies. Consequently, the production of competitive, male-only release cohorts is seen as essential. The RIDL (Release of Insects carrying a Dominant Lethal) system is a transgene-based derivative of SIT, one version of which involves the mass release of insects carrying a female specific lethal transgene (fsRIDL). This thesis describes: 1) the development of fsRIDL olive fly strains and the molecular analysis of transgene insertion and function; 2) the analysis of strain life-history parameters; 3) studies into sexual selection and mating compatibility; 4) a caged proof-of-principle population suppression trial; and, 5) selection dynamics on the fsRIDL trait in caged populations. Olive fly fsRIDL strains were developed with full female-lethal penetrance and repressibility. The lead strain displayed similar life-history and sexual competitiveness traits to those of the wild-type strain from which they were derived. In addition, transgenic males showed photoperiod compatibility and strong sexual competitiveness with field-collected wild olive flies. The feasibility of the fsRIDL approach was demonstrated when repeated male releases caused eradication of caged olive fly populations. Although needing field confirmation, these results suggest that fsRIDL olive fly strains may help to mitigate key problems experienced in previous olive fly SIT trials, and could help form the basis of a renewed effort towards olive fly SIT control
The design and mechanism of synthetic homing endonuclease gene drives
Full text linkWhen humans and insects come into conflict, this can lead to significant ecological and public health challenges. Traditional pest control methods often rely on chemical insecticides, which can adversely affect the environment and non-target species. Genetic biocontrol through the use of gene drive may offer a radically more efficient and species-specific method of addressing pest harm. Synthetic homing gene drives, particularly those based on the CRISPR-Cas9 system, have been the focus of intensive research in recent years. However, there is much we do not understand about the fundamental nuclease processes that mediate gene drive functioning and how these are affected by transgene design. Here, we investigate through computational modelling how unintended nuclease processes affect gene drive performance. We find that certain complex self-limited gene drive designs (daisy-chain gene drives) are especially sensitive to effects that cause separate gene drive elements to segregate prematurely. Through a meta-analysis of experimental gene drive crosses, we evaluate the effect of sex and deposition on different gene drive outcomes. We developed a companion web tool that allows users to find the highest-quality data evaluating specific aspects of gene drive designs and experimental conditions. Lastly, we analyse Aedes aegypti gene drive performance with different nuclease expression patterns. This shows that identical homing gene drives can vary in their underlying mechanism of inheritance bias
Improving transgenic approaches to mosquito population control
Full text linkThe disease vectors Aedes aegypti and Aedes albopictus are serious and growing threats to global health. As vectors of the arboviruses dengue fever and chikungunya, these mosquitoes are responsible for hundreds of millions of cases and thousands of deaths each year. Absent specific treatments or vaccines, effective control of mosquito populations remains the only option for tackling a growing public health challenge. More effective control tools are urgently needed. Recently, a novel approach to pest population control has been developed based on the release of insects carrying a repressible, dominant lethal allele. This approach has achieved dramatic reductions in Ae. aegypti populations in regulated open field experiments. Despite this success, there remains scope to improve upon the current technology. It is proposed that an 'ideal' strain would combine the following features: (i) repressible lethality in late juvenile phases; (ii) a mechanism for removing females at an early developmental stage in the release generation; and (iii) orthogonal expression control mechanisms allowing both these systems to be combined in a single strain. This thesis describes research undertaken in pursuit of a 'next generation' strain. Two novel promoters from putative Osiris genes have been identified which confer a 102-103 – fold up-regulation in transgene expression specific to late pupal stages. One of these 'Osiris' promoters has been used to develop transgenic Aedes aegypti strains. 5 lines showed pupal-specific lethality of 98-100% penetrance, which was repressed in the presence of tetracycline. An Ae. albopictus orthologue of the sex-determining gene doublesex (dsx) has been isolated and characterised and a female-specific expression system developed. Transgenic lines show female-specific expression of a transgene; however, there remains some 'leaky' expression in male insects. Finally, a potential expression control tool based on an auxin-inducible expression system has been investigated. 11 different transgenic lines were developed based on three different construct designs. None showed auxin-inducible expression of a transgene
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