The genetic control of Aedes aegypti

In 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.</p

The Genetic Control of Aedes aegypti

In 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 &Phi;C31-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.</p

Engineered action at a distance: Blood-meal-inducible paralysis in Aedes aegypti.

BACKGROUND:Population suppression through mass-release of Aedes aegypti males carrying dominant-lethal transgenes has been demonstrated in the field. Where population dynamics show negative density-dependence, suppression can be enhanced if lethality occurs after the density-dependent (i.e. larval) stage. Existing molecular tools have limited current examples of such Genetic Pest Management (GPM) systems to achieving this through engineering 'cell-autonomous effectors' i.e. where the expressed deleterious protein is restricted to the cells in which it is expressed-usually under the control of the regulatory elements (e.g. promoter regions) used to build the system. This limits the flexibility of these technologies as regulatory regions with useful spatial, temporal or sex-specific expression patterns may only be employed if the cells they direct expression in are simultaneously sensitive to existing effectors, and also precludes the targeting of extracellular regions such as cell-surface receptors. Expanding the toolset to 'non-cell autonomous' effectors would significantly reduce these limitations. METHODOLOGY/PRINCIPAL FINDINGS:We sought to engineer female-specific, late-acting lethality through employing the Ae. aegypti VitellogeninA1 promoter to drive blood-meal-inducible, fat-body specific expression of tTAV. Initial attempts using pro-apoptotic effectors gave no evident phenotype, potentially due to the lower sensitivity of terminally-differentiated fat-body cells to programmed-death signals. Subsequently, we dissociated the temporal and spatial expression of this system by engineering a novel synthetic effector (Scorpion neurotoxin-TetO-gp67.AaHIT) designed to be secreted out of the tissue in which it was expressed (fat-body) and then affect cells elsewhere (neuro-muscular junctions). This resulted in a striking, temporary-paralysis phenotype after blood-feeding. CONCLUSIONS/SIGNIFICANCE:These results are significant in demonstrating for the first time an engineered 'action at a distance' phenotype in a non-model pest insect. The potential to dissociate temporal and spatial expression patterns of useful endogenous regulatory elements will extend to a variety of other pest insects and effectors

The Potential for a Released Autosomal X-Shredder Becoming a Driving-Y Chromosome and Invasively Suppressing Wild Populations of Malaria Mosquitoes

International audienceSex-ratio distorters based on X-chromosome shredding are more efficient than sterile male releases for population suppression. X-shredding is a form of sex distortion that skews spermatogenesis of XY males towards the preferential transmission of Y-bearing gametes, resulting in a higher fraction of sons than daughters. Strains harboring X-shredders on autosomes were first developed in the malaria mosquito Anopheles gambiae , resulting in strong sex-ratio distortion. Since autosomal X-shredders are transmitted in a Mendelian fashion and can be selected against, their frequency in the population declines once releases are halted. However, unintended transfer of X-shredders to the Y-chromosome could produce an invasive meiotic drive element, that benefits from its biased transmission to the predominant male-biased offspring and its effective shielding from female negative selection. Indeed, linkage to the Y-chromosome of an active X-shredder instigated the development of the nuclease-based X-shredding system. Here, we analyze mechanisms whereby an autosomal X-shredder could become unintentionally Y-linked after release by evaluating the stability of an established X-shredder strain that is being considered for release, exploring its potential for remobilization in laboratory and wild-type genomes of An. gambiae and provide data regarding expression on the mosquito Y-chromosome. Our data suggest that an invasive X-shredder resulting from a post-release movement of such autosomal transgenes onto the Y-chromosome is unlikely

iRMCE in <i>Ae</i>. <i>aegypti</i>.

<p>(a) Schematic diagram of iRMCE in <i>Ae</i>. <i>aegypti</i> and (b) corresponding fluorescent phenotypes in fourth instar <i>Ae</i>. <i>aegypti</i> larvae following OX4714 injections. Black arrows indicate engineered <i>FRT</i> and <i>loxP</i> sites present in donor and acceptor constructs; AmpR represents the plasmid backbone sequences, which includes the ampicillin-resistance gene; grey arrows indicate <i>piggyBac</i> ends. The donor plasmid’s 3xP3-DsRed2 cassette is exchanged for the docking construct’s 3xP3-Amcyan. Larvae are shown under (i) white light, (ii) cyan, and (iii) red excitation light and filters. Images show complete exchange of the 3xP3-AmCyan cassette (OX4476), by integration of 3xP3-DsRed2 (seen in OX4476[OX4714]), and excision of the 3xP3-AmCyan marker (‘excised’ larva). There was reduced expression of the docking cassette’s 3xP3-AmCyan marker following ΦC31-<i>att</i> integration (white arrows: panel (ii), OX4476[OX4714]). Images (i-iii) were taken under the same magnification. Scale bar represents 0.5 mm. White dashed lines outline larvae.</p

Summary of <i>Ae</i>. <i>aegypti</i> OX4312 injections for ΦC31-RMCE.

<p>*All G1 transformants from OX4312 injections into OX4372A resulted in incomplete cassette exchange due to only a single pair of attachment sites recombining. Embryos of this partially recombined line, called ‘OX4372A[OX4312]-incomplete’ were injected with additional ΦC31 recombinase mRNA resulting in a second recombination step in G1 progeny (Efficiency = 18.5%).</p><p>Recombination events (R) are defined as the number of transgenic pools. Efficiency = minimum calculated integration efficiency.</p><p>Summary of <i>Ae</i>. <i>aegypti</i> OX4312 injections for ΦC31-RMCE.</p

iRMCE in <i>P</i>. <i>xylostella</i>.

<p>(a) Schematic diagram of iRMCE in <i>P</i>. <i>xylostella</i> and (b) corresponding fluorescent phenotypes in <i>P</i>. <i>xylostella</i> pupae. Black arrows indicate engineered <i>FRT</i> and <i>loxP</i> sites present in donor and acceptor constructs; AmpR represents the plasmid backbone sequences, which includes the ampicillin-resistance gene; grey arrows indicate <i>piggyBac</i> ends. Pupae are shown under (i) white light, (ii) green excitation light and filters, and (iii) red excitation light and filters. Images (i–iii) were taken under the same magnification. Scale bar repersents 1 mm.</p

Genomic flanking sequences of OX4372 and OX4476 in <i>Ae</i>. <i>aegypti</i>.

<p>The duplicated TTAA <i>piggyBac-</i>insertion site was present in all 5’ and 3’ insertion boundaries (underlined).</p><p>Genomic flanking sequences of OX4372 and OX4476 in <i>Ae</i>. <i>aegypti</i>.</p

Summary of <i>Ae</i>. <i>aegypti</i> and <i>P</i>. <i>xylostella</i> injections for ΦC31-<i>att</i> integration.

<p>Integration events (I) are defined as the number of transgenic pools. Efficiency = minimum calculated integration efficiency.</p><p>Summary of <i>Ae</i>. <i>aegypti</i> and <i>P</i>. <i>xylostella</i> injections for ΦC31-<i>att</i> integration.</p

Summary of <i>Ae</i>. <i>aegypti</i> and <i>P</i>. <i>xylostella</i> injections for Cre and FLP injections.

<p><i>Ae</i>. <i>aegypti</i> (‘<i>Aae</i>’) strains were injected with Cre and FLP in parallel for direct comparison. The number of <i>Ae</i>. <i>aegypti</i> pools with progeny apparently lacking the AmCyan marker (due to weak fluorescence) is shown (‘marker loss’); this was not a problem with the <i>P</i>. <i>xylostella</i> (‘<i>Pxy</i>’) strain so values in this column reflect true marker loss. Excision events (E) were confirmed by PCR and sequencing, and the minimum calculated excision frequencies (Efficiency) are shown as a percentage.</p><p>Summary of <i>Ae</i>. <i>aegypti</i> and <i>P</i>. <i>xylostella</i> injections for Cre and FLP injections.</p