Developing new genomic tools and rapid, automated resistance detection in mosquito larvae
Lead Research Organisation:
Liverpool School of Tropical Medicine
Department Name: Vector Biology
Abstract
Insecticide resistance is a looming threat to malaria and arbovirus control programmes targeting the mosquito vector. Larval resistance is an important phenotype to assess, however, the existing WHO recommended mortality-based larval resistance assay is low-throughput and subject to investigator bias. To address these issues, a novel assay was developed using the Invertebrate Automated Phenotyping Platform (INVAPP) and analysis algorithm (Vectorgon) which provides automated quantification of larval motility after insecticide exposure. Several molecular changes have been observed in mosquitoes displaying resistance to common larvicides. However, their phenotypic characterisation in the absence of other potential resistance mechanisms is largely unexplored. Understanding the potential impact on mosquito control programmes of each mechanism is vital. In susceptible laboratory mosquitoes, upregulation of metabolic enzymes was mimicked using a bipartite GAL4-UAS system. The INVAPP assay was used to assess insecticide susceptibility of transgenic larvae overexpressing individual enzymes - GSTe2, CYP6M2, CYP6P3 and SAP2 - which are implicated in adulticide resistance. A transgenic line expressing CCEae3a (a carboxylesterase which has been identified in Aedes mosquitoes displaying organophosphate (OP) resistance), was found to display resistance to three common OPs in larvae, four-insecticide classes in adults and significant, though moderate, reductions in adult longevity, egg laying, and larval hatch-rate. Using CRISPR-Cas9, a single nucleotide polymorphism (G119S) was introduced in the Acetylcholine esterase 1 (ACE1) gene - the target for OP and carbamate insecticides of susceptible mosquitoes and the resulting phenotypes assessed. The results of this work, highlight important links between adulticide and larvicide resistance mechanisms and the necessity to assess mechanisms individually.
People |
ORCID iD |
Gareth Lycett (Primary Supervisor) |
Publications
Adolfi A
(2018)
Multi-tissue GAL4-mediated gene expression in all Anopheles gambiae life stages using an endogenous polyubiquitin promoter.
in Insect biochemistry and molecular biology
Adolfi A
(2019)
Functional genetic validation of key genes conferring insecticide resistance in the major African malaria vector, Anopheles gambiae.
in Proceedings of the National Academy of Sciences of the United States of America
Adolfi A
(2018)
Opening the toolkit for genetic analysis and control of Anopheles mosquito vectors.
in Current opinion in insect science
Adolfi A
(2021)
Site-Directed fC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria.
in Journal of visualized experiments : JoVE
Buckingham SD
(2021)
Automated phenotyping of mosquito larvae enables high-throughput screening for novel larvicides and offers potential for smartphone-based detection of larval insecticide resistance.
in PLoS neglected tropical diseases
Ingham V
(2019)
A sensory appendage protein protects malaria vectors from pyrethroids
in Nature
Kefi M
(2019)
Two functionally distinct CYP4G genes of Anopheles gambiae contribute to cuticular hydrocarbon biosynthesis.
in Insect biochemistry and molecular biology
Kefi M
(2023)
ABCH2 transporter mediates deltamethrin uptake and toxicity in the malaria vector Anopheles coluzzii.
in PLoS pathogens
Leftwich P
(2023)
A synthetic biology approach to transgene expression
Matthews BJ
(2018)
Improved reference genome of Aedes aegypti informs arbovirus vector control.
in Nature
Partridge FA
(2021)
Actions of Camptothecin Derivatives on Larvae and Adults of the Arboviral Vector Aedes aegypti.
in Molecules (Basel, Switzerland)
Poulton B
(2024)
Aedes aegypti CCEae3A carboxylase expression confers carbamate, organophosphate and limited pyrethroid resistance in a model transgenic mosquito
in PLOS Neglected Tropical Diseases
Poulton BC
(2021)
Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae.
in Journal of visualized experiments : JoVE
Spencer CS
(2018)
Characterisation of Anopheles gambiae heme oxygenase and metalloporphyrin feeding suggests a potential role in reproduction.
in Insect biochemistry and molecular biology
Studentship Projects
Project Reference | Relationship | Related To | Start | End | Student Name |
---|---|---|---|---|---|
MR/P016197/1 | 30/09/2017 | 29/09/2021 | |||
1981052 | Studentship | MR/P016197/1 | 01/10/2017 | 30/09/2021 |
Title | Rebecca Lennon Exhibition at Dilston Grove. London |
Description | Preparing material for a video artwork installation. High resolution video footage and recordings were made of mosquitoes feeding to provide material for an abstract and poetic art film that will provide arresting imagery for projection in the converted church gallery. |
Type Of Art | Artistic/Creative Exhibition |
Year Produced | 2019 |
Impact | The video footage is being prepared for the installation and still images have been provided to the LSTM without copyright to be used for teaching and communication. |
URL | https://www.rebeccalennon.co.uk/ |
Title | Scientific advisor to Christine Poulson to develop ideas for novel |
Description | Novel " An air that kills" features the use of mosquitoes for murder mystery theme for a lab based plot. Advice was given on how mosquitoes are bred, tested for infection and resistance, and consultation on the scientific authenticity of scenarios discussed. |
Type Of Art | Creative Writing |
Year Produced | 2019 |
Impact | Novel published by Lion Fiction worldwide in November 2019. Available from all good book stores and Amazon. currently in the top 1000 all time selling medical thrillers on Amazon |
URL | http://www.christinepoulson.co.uk/ |
Description | MRC P2D |
Amount | £11,700 (GBP) |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 11/2017 |
End | 09/2018 |
Description | P2D |
Amount | £64,000 (GBP) |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 09/2018 |
End | 12/2019 |
Title | High throughput screen for anopheles |
Description | Adapted software to analyse behaviour of Anopheles larvae |
Type Of Material | Technology assay or reagent |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | We have provided proof of principle screens of compounds and selected repurposed pest and agricultural insecticides to identify novel active compounds against mosquitoes |
Description | Rapid, automated insecticide resistance detection in mosquito larvae |
Organisation | University College London |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Our team provided the understanding of mosquito rearing and insecticide resistance and supplied mosquito eggs to the team in London for use in their experiments. Our team generated 4 Anopheles gambiae Gal4-UAS transgenic lines overexpressing enzymes implicated in resistance which have been used in the experiments to date. I have optimized the set up of these transgenic Anopheles larvae for use in the INVAPP imaging system and have begun testing of transgenic larvae using the system. I have also conducted WHO larval assays for comparison against the INVAPP system. |
Collaborator Contribution | The Sattelle group developed the INVAPP system and wrote the analysis programme. They trained me in the methodology and analysis required to use the system. They provided expertise in programming and statistical analysis. |
Impact | None yet. |
Start Year | 2017 |
Description | collaboration with venom unit at LSTM |
Organisation | Liverpool School of Tropical Medicine |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We provided the materials and expertise to examine venom toxicity to mosquitoes |
Collaborator Contribution | Partbers at the venom Unit provided snake venoms for analysis and CeBioscince provided techniques for high throughput assays of toxicity to mosquitoes and worms |
Impact | Manuscripts are in draft and prospective applications for further funding are being discussed |
Start Year | 2019 |
Description | |
IP Reference | |
Protection | Copyrighted (e.g. software) |
Year Protection Granted | |
Licensed | No |
Impact | The technology has been involved in all outputs of this project and collaboration with University College London. |
Description | Engagement with Social Mobility Foundation |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Schools |
Results and Impact | Participation in presentation on the Social Mobility Foundation to discuss mentoring of young people. |
Year(s) Of Engagement Activity | 2019 |
Description | Insectary Tours |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | I regularly lead tours of our insectaries, discussing mosquito behaviour, anatomy, rearing and methods of generating transgenic insects. Usually either individuals or in groups up to 15 at a time (often multiple groups during open days). Participants vary widely (school children, general public, prospective students/research staff, etc) and therefore the outcome of these tours ranges from generating interest in mosquitoes and tropical medicine to attracting new students/staff to the school. |
Year(s) Of Engagement Activity | 2017,2018,2019 |
Description | International Workshop on Pyrethrum: biosynthesis, pest and vector management |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | I gave a 3 minute turbo talk and presented a poster of my research which I then discussed with the other attendees of the workshop. |
Year(s) Of Engagement Activity | 2018 |
URL | http://www.pyrethrum2018.com |
Description | Invited Speaker: Mosquito Research - From Sensory Biology to Vector Control |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Workshop on using research into sensory biology for vector control |
Year(s) Of Engagement Activity | 2019 |
Description | Presentation at Royal Entomological Society PG Forum |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | Presented a 10 minute talk on the novel method of the project at the RES PG Forum which sparked discussions afterwards about the potential of the technique. |
Year(s) Of Engagement Activity | 2020 |
Description | Science museum display |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | We have provided genetically modified mosquitoes to the National Science museum for display in the medicine galleries, to illustrate the potential for disease control. |
Year(s) Of Engagement Activity | 2018 |
Description | Visit to research facility |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | To provide description of the project to heads of Yunnan Institute of Parasitic Diseases to stimulate interests of future collaborations and exchange of students between Institutes |
Year(s) Of Engagement Activity | 2017 |
Description | pyrethrum conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Gareth Lycett: invited talk - Developing a toolkit for genetic analysis and control of malarial vectors |
Year(s) Of Engagement Activity | 2018 |
Description | tour to Annalisa Jenkins |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | Annalisa is a biotech entrepreneur, philanthropist and President of the Court of LSHTM. opportunity to demonstrate the work of the LSTM and project specifics to a key influencer of policy and donor uptake |
Year(s) Of Engagement Activity | 2019 |
Description | turbo talk and poster international conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | PhD student on project Beth Poulton: turbo talk and poster - Rapid, automated insecticide resistance analysis in mosquito larvae |
Year(s) Of Engagement Activity | 2018 |
Description | visit by Finance staff London School of Tropical Medicine and Hygiene |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | Visit by the LSTMH finance dept to provide them with an overview of the research taking place in my lab and vector group to generate cross institute interest in our respective research priorities |
Year(s) Of Engagement Activity | 2019 |
Description | visit by Research England |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | A visit to the insectaries to discuss the work being done in the dept under funding from UKRI, and wider issues of research policy |
Year(s) Of Engagement Activity | 2018 |