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.
 
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