Post Genomic Functional Genetic Analysis of Parasite/Insect Interaction

Lead Research Organisation: Liverpool School of Tropical Medicine
Department Name: Vector Research

Abstract

The recent direction of biological research has focused on sequencing the total DNA content of organisms. This has led to the identification of potentially all the genes encoding for specific proteins present in particular species. What is needed now is to identify the function of these genes, their protein products and the tissues in which they are produced. The most common way to establish gene function is to isolate specific genes in the test tube, modify them and then return them to the organism. These modifications can either cause the organism to over-produce, or conversely, underproduce, this specific protein which in turn causes a specific physiological change that gives vital information on the gene's normal role. In the model organism, the fruit fly, there are numerous systems available to modify gene expression. However, to study important interactions that only occur between parasites and blood sucking insects requires similar methods to be developed for these important carriers of disease. This is particular relevant to mosquitoes which are biting nuisances worldwide and the most important vectors of many human diseases. The general complications of maintenance of blood-feeding insects make the development of efficient modification tools that maximize experimental flexibility a requisite. We will thus adopt the most successful method used in fruit fly research to the mosquito, Anopheles gambiae that is the primary transmitter of the malaria parasite in Africa. Different components of system will be initially tested in the simplified environment of isolated mosquito cells to rapidly identify those that function most efficiently. A selection of the most efficient tools will then be examined directly in mosquitoes to precisely define those that work in the more complex holistic environment of the whole organism. As a proof of principle that the system functions efficiently in mosquitoes we will over-produce and silence specific proteins thought to kill parasites in the insect. Using the flexibility of the system we will determine whether malaria parasite killing is more effective if the protein is produced by the mosquitoes' 'liver', blood cells or its stomach. Although the work is focused on Anopheles mosquitoes and the malaria parasite, the principles and methods should be transferable to other parasites transmitted by mosquitoes and blood sucking insects.

Technical Summary

To study gene function in parasitized haematophagous insects requires the development of methodologies tailored to these non-model organisms. These approaches are urgently needed for mosquitoes, which are biting nuisances worldwide and the most important insect vectors of many human diseases. The labour intensive maintenance of blood-feeding insects makes the development of experimental flexibility in functional genetic characterisation an important requisite. The proposed aim is thus to adopt the binary Gal4/UAS system to A. gambiae mosquitoes, in order to create a panel of driver lines that initially target gene expression to the main tissues involved in parasite/vector interaction, namely the midgut, fat body and hemocytes. These driver lines will then become the references with which to examine parasite/vector interaction in the mosquito through tissue specific transgene expression and gene silencing. Initial experiments will screen alternative Gal4 transactivators and UAS:reporter constructs by transfection of A. gambiae cell lines. Functional transactivators with differential activity will then be assayed in transgenic A. gambiae using a gut-specific promoter and the most sensitive UAS:reporter. The most efficient Gal4 transactivator in transgenic mosquitoes will then be used to develop fat body specific and hemocyte specific driver lines. Using responder lines carrying TEP1 and dsRNA cactus constructs, we will then examine the effect on parasite development when TEP1 is differentially expressed or cactus is specifically silenced in fat body, hemocytes and midgut. The principles developed will be applicable to other hematophagous insects. In addition, the lines developed can be used to study other parasites carried by mosquitoes and further aspects of insect biology related to tissue specific metabolism, including behaviour, insecticide resistance, aging and fertility.

Publications

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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

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Balabanidou V (2016) Cytochrome P450 associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae. in Proceedings of the National Academy of Sciences of the United States of America

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Blagrove MSC (2020) Potential for Zika virus transmission by mosquitoes in temperate climates. in Proceedings. Biological sciences

 
Description The key findings of the project have been:

1) The generation of a graded panel of Gal4 based trans-activators and UAS response elements that can be utilized as a bipartite system to express transgenes at different levels in insect cells.
2) The development of a conditional expression system in transgenic mosquitoes that allows gain and loss of gene function to be examined in vivo for the first time in An. gambiae.
3) The discovery that gene expression was silenced in developing testes when transgenes were placed on the X chromosome, and so supplying valuable evidence for evolutionary theories on demasculinization of X chromosome in a non-model organism.
4) The functionality of the conditional expression system for enhancer trapping in conjunction with recombination mediated cassette exchange.

The vision for the project was to develop tools and resources that could be used by the research community to characterize the many candidate genes that would arise from molecular studies of the An. gambiae genome. New Investigator funding would also establish my position at the LSTM, and provide a centre of excellence for mosquito transgenics in the largest vector research department in the country. In fulfillment of these aims, the transgenic resources developed in this project have so far been used in collaboration with five research teams to generate funds to examine a range of scientific questions that were not possible previously. These have included their use in a) defining sex specific transcriptomes in immature stages, b) examining the role of heam oxygenase in blood meal detoxification, c) analysis of male specific gene function, d) functional analysis of candidate genes for metabolic insecticide resistance and e) proof of principle studies to silence genes involved in cuticular resistance and desiccation tolerance. Furthermore, the resources have also been shared with different research groups that are studying innate immunity to malaria parasite infection, mating compatibility and entrainment of circadian rhythms.

The research initiated in this project has also been crucial to the active participation in two large European/African Research Networks funded through EU FP7. In both Networks, I have been leader of the work packages concerned with functional genetic analysis. It has also led to participation in the Insect Genetic Technologies Research Co-ordination Network that brings together researchers from many different fields to share knowledge, skills and training opportunities.
The latest work that has stemmed from this funding has been the identification of two key genes that regulate cuticle structure and thus insecticide penetration.
Exploitation Route The research is aimed towards alleviating the burden of malaria in the longer term. The tools and resources produced are being used to define factors that influence malaria transmission. As outlined above, the transgenic resources developed in this project have already being taken up by a number of academic partners for collaborative and individual research projects. To facilitate future work, we have made the transgenic lines available at the MR4 repository for long term maintenance and free distribution to researchers.

The tools and resources can also be adapted to other disease vectors and agricultural insects. An example of the latter, is our ongoing assistance of partners at Rothamstead Research in the development of transgenic technologies in Plutella xylostella.

The resources produced may also prove invaluable for chemical and biological control of mosquitoes that will interest the non-academic sectors. Research with these tools is likely to produce modified mosquitoes that have altered insecticide metabolism and uptake. These will naturally feed into novel insecticide screening pipelines that have been developed at the LSTM though partnership between the Liverpool Insect Testing Establishment and the Innovative Vector Control Consortium. This partnership enables large chemical companies to screen novel compounds destined for public health use on resistant mosquito populations.

GM mosquitoes are also being trailed currently for the control of dengue in several countries with the products of a UK company. Similar work at UK and International research institutes is developing related approaches for An. gambiae control. The resources developed in this project will assist in the identification of appropriate targets for genetic control both from within the department, our collaborators and others taking up these advanced tools.

The GM are currently being used to screen novel insecticides, and have been made available to the IVCC for their further distribution and utilisation.
Sectors Agriculture, Food and Drink,Chemicals,Pharmaceuticals and Medical Biotechnology

 
Description The New Investigator Research Project initiated the development of a centre of excellence for transgenic technology at the LSTM. The project focus was on basic research to generate research tools to examine gene function in malaria transmitting mosquitoes. These tools have been taken up by many academic partners described elsewhere to further their research goals. However, the long aim is to use this knowledge to relieve the burden of disease in endemic countries through the development of novel control methods and products. To date the major impacts have been educational through public engagement and research and skills training . The transgenic mosquitoes feature heavily on regular visits from schools, the military, chemical industry partners, government and non-governmental officials. Of particular note was the visit of the Minister of Universities and Science, David Willetts MP to discuss the research and its impact. Members of the Lycett lab also participate in outreach programs with the Liverpool World museum to display mosquitoes and other vectors of disease. This stimulates and feeds a great deal of interest in bugs, disease and genetic manipulation. Funding has also been sought to the Wellcome Trust for their People Award for public dissemination of information through a multimedia presentation of "Mosquito watch: there is more to a mosquito than meets the eye." The LSTM also has an active communications group that interacts with the media to stimulate interest and explain the findings of the research. In the last few years, the transgenic mosquitoes have featured in a number of TV broadcasts, including a BBC breakfast news bulletin (31/7/14), Help! I caught it Abroad 2 (9/9/10) and the discovery channel series, Bugs Bites and Parasites (August/Sept 2013). This exposure has also stimulated media enquiries to provide expert commentary on breaking news concerned with mosquitoes and their genetic control. More recently (early 2106) the transgenic facility was visited by George Osborne and Bill Gates as part of their unveiling of the Ross Fund to provide research funds for malaria and neglected tropical diseases over the next years From an economic perspective, the establishment of the transgenic facility has also provided opportunities for skills training of M.Sc and B.Sc. students that have subsequently moved into both industrial and academic positions. In addition, the facility has given expert training to technicians that have been employed by the Liverpool Insect Testing Establishment (LITE); a spin off facility that screens new insecticide products produced by commercial partners, including Bayer, Syngenta and Sumitomo, against insecticide sensitive and resistant populations of mosquitoes. The work has now progressed whereby the GM mosquitoes produced are being used to screen new compounds for efficacy against the resistant mosquitoes generated. This commercial application is being exploited by IVCC and their corporate partners to introduce new compounds in malaria vector control.
First Year Of Impact 2016
Sector Education,Government, Democracy and Justice,Manufacturing, including Industrial Biotechology,Culture, Heritage, Museums and Collections
Impact Types Societal,Policy & public services

 
Description FP7 Health
Amount € 12,000,000 (EUR)
Funding ID 265660 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 02/2011 
End 01/2016
 
Description Focused research project
Amount € 2,900,000 (EUR)
Funding ID 223736 
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 12/2008 
End 06/2013
 
Description IVCC project award
Amount £18,000 (GBP)
Organisation IVCC 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2018 
End 07/2018
 
Description MRC iCASE Studentship
Amount £120,000 (GBP)
Funding ID MR/P016197/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2017 
End 09/2021
 
Description Research Project Grant
Amount £202,000 (GBP)
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2012 
End 09/2015
 
Description Responsive mode project
Amount £267,710 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2009 
End 08/2012
 
Title GAL4 UAS system developed for An gambiae 
Description We have developed highly efficient systems for functional genetic analysis in mosquitoes. Most notably, the GAL4 system and recombination mediated cassette exchange in An gambiae mosquitoes. These are suitable for conditional expression of transgenes in this vector of malaria, as well as the development of enhancer trapping for the first time in this species. The Gal4 lines developed have been deposited at MR4 facility for distribution to researchers 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact The research tool has opened up several avenues of research that were impossible previously, both within the department and for other groups. Most notably, we are using the GAL4 UAS system to examine the in vivo roles of genes involved in insecticide resistance, parasite development and most recently the knockdown of genes regulating cuticular hydrocarbon and pheromone production. These are the first studies that will genetically modify the behaviour and desiccation tolerance of mosquitoes. In addition, the work has provided a number of transgenic lines suitable for other purposes that we have provided to collaborators that are currently being exploited, including the production of fluorescently tagged mosquitoes that can be used for sexing young larvae that has enabled sex specific sequencing of immature stages for the first time. In addition, we have tagged sperm so that mating success can be rapidly followed. Similarly, ubiquitously tagged lines are being used to develop novel RNAi technology for the mosquito. 
URL http://www.mr4.org/MR4ReagentsSearch/livingMosquitoes/MRA-1167.aspx
 
Title RCME 
Description We have shown that Recombination Mediated Cassette Exchange is highly efficient in mosquitoes using the phiC31 integrase, and used this tool in combination with the Gal4 system to develop enhancer trapping in An. gambiae. The tool allows any number of transgenes to be swopped into identical chromosomal sites to compare their activities without the problems of position effect variagation and insertion of unwanted plasmid sequences. 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact We have developed enhancer trapping in An. gambiae with these tools which has allowed us to target gene expression to cells regulating pheromone and hydrocarbon production in a non drosophilid insect for the first time. 
 
Title Tissue specifc gene knockdown 
Description Tissue specific gene knockdown tools developed in Anopheles mosquitoes to examine cuticle structure 
Type Of Material Biological samples 
Year Produced 2015 
Provided To Others? Yes  
Impact Tool has been used for the first time to demonstrate metabolomics on single mosquitoes 
 
Title mosquito sexing tool 
Description X linked flourescent tag in anopheles allows sex specific analysis of transcriptomics and proteomics 
Type Of Material Biological samples 
Year Produced 2010 
Provided To Others? Yes  
Impact Transcriptomic analysis sex specific expression of early mosquito larvae, identifying dosage compensation and Y linked sex determination Publication submitted to Science and recent publication in genome Biology 
 
Description Analysis of gene function in Aedes aegypti 
Organisation University of Victoria
Country Australia 
Sector Academic/University 
PI Contribution We provide expertise in mosquito trasngenesis to define experiments to analyse gene function of immune genes in Aedes aegypti
Collaborator Contribution Partners have provided ecological and evolutionary analysis of gene diversity and selection to define candidate genes under immune selectionin field populations.
Impact Drafting application for joint research funding
Start Year 2016
 
Description Arboviral Infection Studies 
Organisation University of Liverpool
Department Institute of Infection and Global Health
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided the background knowledge, infrastructure and biological safety expertise to set up the first institute in the UK to examine arboviral infections in mosquitoes.
Collaborator Contribution The partners have developed projects and PI funding to follow arboviral infections in wild caught britsh mosquitoes. The work has since extend to Dengue and West Nile Virus analysis in exotic mosquitoes, and current applications look at Zika infections
Impact Submitted joint publication. BBSRC funding
Start Year 2011
 
Description Infection of field Anopheles funestus with plasmodium falciparum 
Organisation Ozics
Country Finland 
Sector Private 
PI Contribution I am sponsor for a Wellcome Inertemediate fellowship based in Cameroon. I provide expertise in RNAi and plasmodium infection analysis to the study
Collaborator Contribution Collaborators are carrying out infection studies on wild caught mosquitoes to define their infectibility for the first time with natural field isolates of plasmodium
Impact Wellcome Training Fellowship Publication in Draft
Start Year 2013
 
Description Metabolomics of mosquito gene knockouts 
Organisation University of Liverpool
Department Institute of Integrative Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided gene knockout samples to be examined by NMR to determine the effect on the metabolome of gene silencing. These have provided rare material for PhD project in the dept.
Collaborator Contribution The partners have included their samples in a PhD project to examine the feasibility of single mosquito specimen metabolomics. They provide all the material for NMR analysis and characterisation.
Impact Publication in draft. Molecular genetics combined with systems biology, NMR.
Start Year 2015
 
Description Plasmodium infection of transgenic lines 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Transgenic mosquito lines were supplied to Imperial College to examine plasmodium infection status. Discussion of future collaborative work to expand on the success of the BBSRC work for further funding on related projects. We also identified the that X linked silencing was occurring in the transgenic mosquitoes we had generated. Discussions of this with the Imperial group indicated that they had similar results in entirely separate project, joint results were submitted as a co-publication.
Collaborator Contribution The Partners examined the lines for infection status. Collaborative discussion for joint research to follow up this project which resulted in collaborative grant from EU FP7 with multiple European and African partners. Similar results from related projects were combined to produce a joint publication.
Impact X linked gene silencing was published in BMC Evolutionary Biology Award of Transmalariabloc grant from EU
Start Year 2008
 
Description Prospective liability of novel and repurposed compounds to metabolic resistance in An. gambiae 
Organisation IVCC
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Produced genetically modified mosquitoes that overexpress specific P450s that confer resistance to current insecticides
Collaborator Contribution Partners provide the interface to collaborate effective with multiple industrial partners to produce and test novel compounds to control vector populations. As a partnership we are testing whether these novel compounds are susceptible to metabolism by mosquitoes, and thus likely to be effective in the field. The work also tests whether related re-purposed compounds, likely to be cheaper, are as efficient as the novel compounds
Impact Interim report on the first phase of compound testing. Application for further funds to expand the work to other GM mosquito lines,
Start Year 2017
 
Description Prospective liability of novel and repurposed compounds to metabolic resistance in An. gambiae (2) 
Organisation Liverpool School of Tropical Medicine
Department Liverpool Insect Testing Establishment
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide screened and sexed genetically modified mosquitoes to the facility so that they can test their resistance characteristics against novel and repurposed compounds
Collaborator Contribution The LITE facility commercially tests active ingredients and compounds for toxicity and repellency against a variety of mosquito species. It is supported by the IVCC in their quest to discover new compounds for malaria control.
Impact First set of assays performed that have produced an interim report on relative sensitivity to compounds.
Start Year 2017
 
Description Prospective liability of novel and repurposed compounds to metabolic resistance in An. gambiae (3) 
Organisation CE Bioscience Ltd
Country United Kingdom 
Sector Private 
PI Contribution We lead a research project examining the genetics of insecticide resistance in msoquitoes. We perform functional genetic modification to examine the role of different gene families in resistacne phenotypes, in order to develop methods and compounds to overcome this resistance.
Collaborator Contribution The partners have developed a high throughput rapid screen of larval susceptibility to compounds based on image analysis of multiwell plates of larvae to monitor behaviour and death.
Impact Successful application for an MRC iCASE Studentship to develop the system further. The collaboration is multidisciplinary involving biological, physiochemcal and computational analysis to provide both qualitative and quantitative analysis.
Start Year 2017
 
Description Provision of X linked transgenic mosquitoes 
Organisation Liverpool School of Tropical Medicine
Department Department of Vector Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Transgenic mosquitoes were supplied that enabled sexing of early mosquito stages. Following on from this collaboration, I am Institute supervisor for PhD student based in Pirbright examining sex determination in mosquitoes.
Collaborator Contribution Partners used these mosquitoes as a basis for successful grant application to examine genes regulating sex determination and differentiation in An. gambiae.
Impact PhD Studentship at Pirbright Vectorbase grant for 'Ultra-deep exploration of transcription in Anopheles gambiae' 2011-2013 Manuscript Dosage Compensation in the African Malaria Mosquito Anopheles gambiae. Rose G, Krzywinska E, Kim J, Revuelta L, Ferretti L, Krzywinski J. Genome Biol Evol. 2016 Jan 18;8(2):411-25. doi: 10.1093/gbe/evw004.
Start Year 2011
 
Description Provision of transgenic mosquitoes for RNAi screendevelopment 
Organisation Kansas State University
Country United States 
Sector Academic/University 
PI Contribution Agreement to supply fluorescent transgenic mosquitoes for a project application to develop novel RNAi tools
Collaborator Contribution The collaborators are applying for funds to develop novel RNAi tools for the mosquito and require the fluorescently tagged mosquitoes we have developed in the BBSRC project
Impact Application for R21 funds from NIH
Start Year 2014
 
Description Supply of mosquitoes having fluorescently tagged sperm 
Organisation University of Perugia
Country Italy 
Sector Academic/University 
PI Contribution Supplied mosquitoes with flourescently tagged sperm.
Collaborator Contribution Partners need a rapid way to screen for mated female mosquitoes, and the tagged sperm mosquitoes we developed in the BBSRC project are ideal for this
Impact None as yet
Start Year 2014
 
Description supply of Gal4 mosquitoes 
Organisation Imperial College London
Department Imperial College Trust
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Provide Gal4 and UAS mosquitoes for their experiments of gut signalling
Collaborator Contribution Their work is expanding from drosophila in mosquitoes, and the lines we produced from BBSRC funding will facilitate this.
Impact just beginning
Start Year 2016
 
Description Media Interviews 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Various press releases and interviews have occured, mainly relating to a media story about mosquito borne disease transmission in the news where they require an expert in the field to comment

Contacts made within the BBC that now regularly look to the LSTM for science/health commentaries
Year(s) Of Engagement Activity 2010,2011,2012,2014,2015
URL http://www.bbc.com/news/science-environment-10654599
 
Description Mosquito conference (Crete) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Conference talk to provide latest research stemming from BBSRC new investigator funding. Discussion and collaboration afterwards.
Year(s) Of Engagement Activity 2016
 
Description National Museums Liverpool 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Exhibitions generated interest and questions from all ages

Asked for future commitments to the scheme
Year(s) Of Engagement Activity 2010,2012,2014
URL http://www.nationalinsectweek.co.uk/events/creepy_crawly_week
 
Description UK Gov visist 
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 George Osborne and Bill Gates visited the Group to discuss our work and advertise through the media the release of funds for malaria and neglected disease control.
Visit reported on local and National TV and papers.
Year(s) Of Engagement Activity 2016
URL http://www.lstmed.ac.uk/news-events/news/lstm-hosts-the-formal-announcement-of-the-ross-fund
 
Description Visits 
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 Schools
Results and Impact The exhibition stimulates questions related to many aspects of malaria and disease control and the use of GM mosquitoes.

Secretary of State for Science and Universities became aware of the breadth of work undertaken at the LSTM and the state of the art tools that the project has produced.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015