Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' ...

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...

Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control ...

Liverpool School of Tropical Medicine

University of California

University of California, Irvine

liverpool school of tropical medicine

The ability to manipulate the Anopheles gambiae genome and alter gene expression effectively and reproducibly is a prerequisite for functional genetic analysis and for the development of novel control strategies in this important disease vector. However, in vivo transgenic analysis in mosquitoes is limited by the lack of promoters active ubiquitously. To address this, we used the GAL4/UAS system to investigate the promoter of the An. gambiae Polyubiquitin-c (PUBc) gene and demonstrated its ability to drive expression in mosquito cell culture before incorporation into An. gambiae transgenic driver lines. To generate such lines, piggyBac-mediated insertion was used to identify genomic regions able to sustain widespread expression and to create φC31 docking lines at these permissive sites. Patterns of expression induced by PUBc-GAL4 drivers carrying single intergenic insertions were assessed by crossing with a novel responder UAS-mCD8:mCherry line that was created by φC31-mediated integration. Amongst the drivers created at single, unique chromosomal integration loci, two were isolated that induced differential expression levels in a similar multiple-tissue spatial pattern throughout the mosquito life cycle. This work expands the tools available for An. gambiae functional analysis by providing a novel promoter for investigating phenotypes resulting from widespread multi-tissue expression, as well as identifying and tagging genomic sites that sustain broad transcriptional activity.

Multi-tissue GAL4-mediated gene expression in all Anopheles gambiae life stages using an endogenous polyubiquitin promoter.

Anopheles is the only genus of mosquitoes that transmit human malaria and consequently the focus of large scale genome and transcriptome-wide association studies. Genetic tools to define the function of the candidate genes arising from these analyses are vital. Moreover, genome editing offers the potential to modify Anopheles population structure at local and global scale to provide complementary tools towards the ultimate goal of malaria elimination. Major breakthroughs in Anopheles genetic analysis came with the development of germline transformation and RNA interference technology. Yet, the field has been revolutionised again by precise genome editing now possible through site-specific nucleases. Here we review the components of the current genetic toolkit available to study Anopheles, focusing particularly on how these technical advances are used to gain insight into malaria transmission and the design of genetic methods to control Anopheles vectors.

Opening the toolkit for genetic analysis and control of Anopheles mosquito vectors.

Resistance in  to members of all 4 major classes (pyrethroids, carbamates, organochlorines, and organophosphates) of public health insecticides limits effective control of malaria transmission in Africa. Increase in expression of detoxifying enzymes has been associated with insecticide resistance, but their direct functional validation in  is still lacking. Here, we perform transgenic analysis using the GAL4/UAS system to examine insecticide resistance phenotypes conferred by increased expression of the 3 genes-, , and -most often found up-regulated in resistant  We report evidence in  that organophosphate and organochlorine resistance is conferred by overexpression of GSTE2 in a broad tissue profile. Pyrethroid and carbamate resistance is bestowed by similar  overexpression, and  confers only pyrethroid resistance when overexpressed in the same tissues. Conversely, such  overexpression increases susceptibility to the organophosphate malathion, presumably due to conversion to the more toxic metabolite, malaoxon. No resistant phenotypes are conferred when either  gene overexpression is restricted to the midgut or oenocytes, indicating that neither tissue is involved in insecticide resistance mediated by the candidate P450s examined. Validation of genes conferring resistance provides markers to guide control strategies, and the observed negative cross-resistance due to  gives credence to proposed dual-insecticide strategies to overcome pyrethroid resistance. These transgenic -resistant lines are being used to test the "resistance-breaking" efficacy of active compounds early in their development.

Functional genetic validation of key genes conferring insecticide resistance in the major African malaria vector, .

Fenazaquin, pyridaben, tolfenpyrad and fenpyroximate are Complex I inhibitors offering a new mode of action for insecticidal malaria vector control. However, extended exposure to pyrethroid based products such as long-lasting insecticidal nets (LLINs) has created mosquito populations that are largely pyrethroid-resistant, often with elevated levels of P450s that can metabolise and neutralise diverse substrates. To assess cross-resistance liabilities of the Complex I inhibitors, we profiled their susceptibility to metabolism by P450s associated with pyrethroid resistance in Anopheles gambiae (CYPs 6M2, 6P3, 6P4, 6P5, 9J5, 9K1, 6Z2) and An. funestus (CYP6P9a). All compounds were highly susceptible. Transgenic An. gambiae overexpressing CYP6M2 or CYP6P3 showed reduced mortality when exposed to fenpyroximate and tolfenpyrad. Mortality from fenpyroximate was also reduced in pyrethroid-resistant strains of An. gambiae (VK7 2014 and Tiassalé 13) and An. funestus (FUMOZ-R). P450 inhibitor piperonyl butoxide (PBO) significantly enhanced the efficacy of fenpyroximate and tolfenpyrad, fully restoring mortality in fenpyroximate-exposed FUMOZ-R. Overall, results suggest that in vivo and in vitro assays are a useful guide in the development of new vector control products, and that the Complex I inhibitors tested are susceptible to metabolic cross-resistance and may lack efficacy in controlling pyrethroid resistant mosquitoes.

New insecticide screening platforms indicate that Mitochondrial Complex I inhibitors are susceptible to cross-resistance by mosquito P450s that metabolise pyrethroids.

Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission. These technologies include population suppression approaches, genetic analogs of insecticidal techniques that reduce the number of insect vectors, and population modification (replacement/alteration) approaches, which interfere with competence to transmit pathogens. Here, we develop a recoded gene-drive rescue system for population modification of the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and rare functional resistant alleles do not prevent drive invasion. Small cage trials show that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios.

Adriana Adolfi

Department of Vector Biology,
Vector Biology Department

Liverpool School of Tropical Medicine

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes

Adriana Adolfi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Vector Biology,Vector Biology Department

Liverpool School of Tropical Medicine

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes

Department of Vector Biology,
Vector Biology Department