using-the-gal4-uas-system-for-functional-genetics-in-anopheles-gambiae

Using the GAL4-UAS System for Functional Genetics in <em>Anopheles gambiae</em>

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' ...

Liverpool School of Tropical Medicine

liverpool school of tropical medicine

Functional genetic analysis in Anopheles gambiae would be greatly improved by the development of a binary expression system, which would allow the more rapid and flexible characterisation of genes influencing disease transmission, including those involved in insecticide resistance, parasite interaction, host and mate seeking behaviour. The Gal4-UAS system, widely used in Drosophila melanogaster functional genetics, has been significantly modified to achieve robust application in several different species. Towards this end, previous work generated a series of modified Gal4 constructs that were up to 20 fold more active than the native gene in An. gambiae cells. To examine the Gal4-UAS system in vivo, transgenic An. gambiae driver lines carrying a modified Gal4 gene under the control of the carboxypeptidase promoter, and responder lines carrying UAS regulated luciferase and eYFP reporter genes have been created. Crossing of the Gal4 and UAS lines resulted in progeny that expressed both reporters in the expected midgut specific pattern. Although there was minor variation in reporter gene activity between the different crosses examined, the tissue specific expression pattern was consistent regardless of the genomic location of the transgene cassettes. The results show that the modified Gal4-UAS system can be used to successfully activate expression of transgenes in a robust and tissue specific manner in Anopheles gambiae. The midgut driver and dual reporter responder constructs are the first to be developed and tested successfully in transgenic An. gambiae and provide the basis for further advancement of the system in this and other insect species.

Development of the bi-partite Gal4-UAS system in the African malaria mosquito, Anopheles gambiae.

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.

Gareth John Lycett

Department of Vector Biology

Liverpool School of Tropical Medicine

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