| Abstract | Insecticides targeting mosquitoes in the Anopheles genus have been highly successful in malaria control. However, progress is threatened by the evolution of insecticide resistance. Resistance is currently monitored using bioassays in which live mosquitoes are exposed to insecticides. Assays are labour intensive and difficult to compare between different laboratories or even days due to high sensitivity to external factors such as temperature. Molecular surveillance of mosquito vectors such as genomic surveillance for resistance associated mutations could provide key information to decision makers selecting vector control strategies, with higher reproducibility and richer information from each mosquito assayed. However, currently available genomic markers mainly assay target site resistance. This is partly due to the greater challenge of developing markers to assay for other resistance mechanisms such as metabolic resistance or cuticular resistance. Much of the variation in metabolic and cuticular insecticide resistance phenotypes is still unexplained by known genomic variants, and where the variants are known, different genetic variants at the same locus can lead to resistance. The evolution of gene expression plays a key role in mosquito insecticide resistance, with genes involved in metabolic resistance and cuticle repeatedly implicated across multiple resistant populations. Gene transcription is regulated both in cis by linked genomic sequences called cis-regulatory modules (CRM) and in trans by diffusible factors such as transcription factors that respond to the cellular environment. Mutations in CRM underlying differential gene regulation of resistance associated genes are good candidates for genomic surveillance of metabolic and cuticular resistance. Alternatively, gene expression could instead be monitored directly (expression markers). It is important to consider the balance of cis and trans regulation of expression markers: as cis-regulation is not impacted by environmental conditions, primarily cis-regulated gene expression would produce a more reproducible signal of resistance than primarily trans-regulated genes. Previous research at LSTM (Dyer et al 2024) has established the use of allele specific expression to study cis regulation of transcription in Anopheles gambiae. Building on this approach and benefiting from funding from the Royal Society, aim 1 of this project is to characterize cis and trans regulation of both constitutive and insecticide induced expression, producing a shortlist of expression markers for molecular surveillance. Data from the Malaria Vector Genome Observatory (https://www.malariagen.net/vobs/) will then be used to identify variants in the putative CRMs regulating these genes in wild Anopheles populations. Aim 2 is to develop CRM variants into viable markers for molecular surveillance. Functional validation of genetic variants’ roles in insecticide resistance in transgenic mosquitoes is a highly labour-intensive process, creating a bottleneck in screening these variants as molecular surveillance markers. This project addresses this bottleneck by developing high throughput cell culture-based assays of CRM activity. Working closely with LSTM experts in activity-based probes and “organ on a chip”, assays will be designed to be accurately representative of insecticide resistance relevant tissues in the mosquito. This method will then be used to shortlist candidate CRM variants both from aim 1 and from the published literature for future validation in transgenic mosquitoes and use in molecular surveillance. |
| Where does this project lie in the translational pathway? | T1 - Basic Research |
| Expected Outputs | • High impact publications on the regulation of gene expression during the evolution of insecticide resistance and cell based assays for high-throughput screening of molecular surveillance candidates • Technical skills in a wide range of quantitative bioinformatics analysis and integration of large datasets • Impact: cell culture-based assays for high throughput functional validation of genetic variants in cis regulatory modules for molecular surveillance. These assays could easily be adapted for more general functional validation prior to costly mosquito experiments, such as investigating the effects of multiple nonsynonymous mutations in protein-coding genes. This advance would be tranaformative for the speed and effiociency of future research in this field, accelerating the pathway from discovery to product (T1 to T2). |
| Training Opportunities | Most of the training in the technical areas of this PhD are available in person at LSTM, taking advantage of the wealth of technical expertise in the department of vector biology. The supervisors will also support the student to identify suitable external training courses as necessary to develop skills throughout the project which may be online or in person courses or visits to other laboratories to learn specific techniques. The main areas from training are listed below: • Training in insectary skills including mosquito rearing, genetic crosses, bioassays of insecticide toxicity • Laboratory skills including molecular biology, cell culture • Training in generation and analysis and integration of large ‘omics datasets • Training in presenting research including at national and international conferences • Mentorship in writing and publishing high impact papers |
| Skills Required | This project is suited to a highly motivated student who is both curious about vector biology and passionate about creating better tools for malaria control. Skills: problem solving, clear communication, taking initiative Experience: experience of biomedical laboratory research and/or coding and data analysis would be beneficial Aptitudes: the student should be numerate and able to combine creativity with a systematic approach to research |
|
Key Publications associated with this project |
1. Dyer NA, Lucas ER, Nagi SC, McDermott DP, Brenas JH, Miles A, et al. Mechanisms of transcriptional regulation in Anopheles gambiae revealed by allele-specific expression. Proceedings Biological sciences / The Royal Society. 2024;291(2031):20241142. https://doi.org/10.1098/rspb.2024.1142 |
| 2. Lucas ER, Nagi SC, Egyir-Yawson A, Essandoh J, Dadzie S, Chabi J, et al. Genome-wide association studies reveal novel loci associated with pyrethroid and organophosphate resistance in Anopheles gambiae and Anopheles coluzzii. Nature communications. 2023;14(1):4946. https://doi.org/10.1038/s41467-023-40693-0 | |
| 3. Ingham VA, Wagstaff S, Ranson H. Transcriptomic meta-signatures identified in Anopheles gambiae populations reveal previously undetected insecticide resistance mechanisms. Nature communications. 2018;9(1):5282. https://doi.org/10.1038/s41467-018-07615-x | |
| 4. Holm I, Nardini L, Pain A, Bischoff E, Anderson CE, Zongo S, et al. Comprehensive Genomic Discovery of Non-Coding Transcriptional Enhancers in the African Malaria Vector Anopheles coluzzii. Frontiers in genetics. 2021;12:785934. https://doi.org/10.3389/fgene.2021.785934 | |
| 5. Ismail HM, O'Neill PM, Hong DW, Finn RD, Henderson CJ, Wright AT, et al. Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(49):19766-71. https://doi.org/10.1073/pnas.1320185110 |