Publications by authors named "Adriana Adolfi"

9 Publications

  • Page 1 of 1

Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes.

J Vis Exp 2021 05 1(171). Epub 2021 May 1.

Department of Microbiology & Molecular Genetics, University of California, Irvine; Department of Molecular Biology & Biochemistry, University of California, Irvine;

Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination.
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http://dx.doi.org/10.3791/62588DOI Listing
May 2021

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

J Vis Exp 2021 04 15(170). Epub 2021 Apr 15.

Department of Vector Biology, Liverpool School of Tropical Medicine;

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' lines that express the yeast transcription factor GAL4 in a tissue specific manner, with transgenic 'responder' lines carrying a candidate gene/RNA interference construct whose expression is controlled by Upstream Activation Sequences (UAS) that bind GAL4. In the ensuing progeny, the gene or silencing construct is thus expressed in a prescribed spatiotemporal manner, enabling the resultant phenotypes to be assayed and gene function inferred. The binary system enables flexibility in experimental approaches to screen phenotypes generated by transgene expression in multiple tissue-specific patterns, even if severe fitness costs are induced. We have adapted this system for Anopheles gambiae, the principal malaria vector in Africa. In this article, we provide some of the common procedures used during GAL4-UAS analysis. We describe the An. gambiae GAL4-UAS lines already generated, as well as the cloning of new responder constructs for upregulation and RNAi knockdown. We specify a step by step guide for sexing of mosquito pupae to establish genetic crosses, that also includes screening progeny to follow inheritance of fluorescent gene markers that tag the driver and responder insertions. We also present a protocol for clearing An. gambiae embryos to study embryonic development. Finally, we introduce potential adaptions of the method to generate driver lines through CRISPR/Cas9 insertion of GAL4 downstream of target genes.
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http://dx.doi.org/10.3791/62131DOI Listing
April 2021

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

J Vis Exp 2021 02 2(168). Epub 2021 Feb 2.

Department of Microbiology & Molecular Genetics, University of California; Department of Molecular Biology & Biochemistry, University of California.

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the φC31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces φC31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the φC31 integrase. We report two representative results of φC31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. φC31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the φC31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.
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http://dx.doi.org/10.3791/62146DOI Listing
February 2021

Hidden genomic features of an invasive malaria vector, Anopheles stephensi, revealed by a chromosome-level genome assembly.

BMC Biol 2021 02 10;19(1):28. Epub 2021 Feb 10.

Department of Ecology and Evolutionary Biology, University of California, Irvine, CA, 92697, USA.

Background: The mosquito Anopheles stephensi is a vector of urban malaria in Asia that recently invaded Africa. Studying the genetic basis of vectorial capacity and engineering genetic interventions are both impeded by limitations of a vector's genome assembly. The existing assemblies of An. stephensi are draft-quality and contain thousands of sequence gaps, potentially missing genetic elements important for its biology and evolution.

Results: To access previously intractable genomic regions, we generated a reference-grade genome assembly and full transcript annotations that achieve a new standard for reference genomes of disease vectors. Here, we report novel species-specific transposable element (TE) families and insertions in functional genetic elements, demonstrating the widespread role of TEs in genome evolution and phenotypic variation. We discovered 29 previously hidden members of insecticide resistance genes, uncovering new candidate genetic elements for the widespread insecticide resistance observed in An. stephensi. We identified 2.4 Mb of the Y chromosome and seven new male-linked gene candidates, representing the most extensive coverage of the Y chromosome in any mosquito. By tracking full-length mRNA for > 15 days following blood feeding, we discover distinct roles of previously uncharacterized genes in blood metabolism and female reproduction. The Y-linked heterochromatin landscape reveals extensive accumulation of long-terminal repeat retrotransposons throughout the evolution and degeneration of this chromosome. Finally, we identify a novel Y-linked putative transcription factor that is expressed constitutively throughout male development and adulthood, suggesting an important role.

Conclusion: Collectively, these results and resources underscore the significance of previously hidden genomic elements in the biology of malaria mosquitoes and will accelerate the development of genetic control strategies of malaria transmission.
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http://dx.doi.org/10.1186/s12915-021-00963-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876825PMC
February 2021

Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi.

Nat Commun 2020 11 3;11(1):5553. Epub 2020 Nov 3.

Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, 92697-3900, USA.

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.
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http://dx.doi.org/10.1038/s41467-020-19426-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609566PMC
November 2020

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

Sci Rep 2020 10 1;10(1):16232. Epub 2020 Oct 1.

Vector Biology Department, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK.

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.
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http://dx.doi.org/10.1038/s41598-020-73267-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530702PMC
October 2020

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

Proc Natl Acad Sci U S A 2019 12 4;116(51):25764-25772. Epub 2019 Dec 4.

Vector Biology Department, Liverpool School of Tropical Medicine, L3 5QA Liverpool, United Kingdom

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.
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http://dx.doi.org/10.1073/pnas.1914633116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926047PMC
December 2019

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

Curr Opin Insect Sci 2018 12 26;30:8-18. Epub 2018 Jul 26.

Vector Biology Department, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK.

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.
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http://dx.doi.org/10.1016/j.cois.2018.07.014DOI Listing
December 2018

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

Insect Biochem Mol Biol 2018 05 22;96:1-9. Epub 2018 Mar 22.

Liverpool School of Tropical Medicine, Vector Biology Department, Liverpool, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ibmb.2018.03.005DOI Listing
May 2018
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