Deciphering molecular basis of mosquito development and vectorial capacity to facilitate novel approaches to vector-borne disease control

Lead Research Organisation: The Pirbright Institute
Department Name: UNLISTED

Abstract

Insect-borne diseases affecting livestock and man constitute a major international socio-economic and public health problem. Understanding the basic biology of insect vectors is essential for the development of more effective approaches to control these diseases. Our current work concentrates on genes involved in mosquito sex determination and spermatogenesis pathways. We use state of the art methods of transcriptome profiling and comparative genomics to identify genes vital in these developmental processes. Our findings are further validated by qRT-PCR and functional analyses involving RNA in situ hybridization and in vivo knock-down or overexpression of individual genes in transgenic mosquitoes. Interfering with the expression of sex determination genes should lead to female-specific embryonic lethality (recently documented by our group) or masculinization. Spermatogenesis genes with no discernible homologues in non-mosquito taxa represent highly promising targets to cause selective mosquito-specific male sterility. Information on genes from both pathways could be utilized in transgenic technology to conditionally eliminate females and efficiently produce sterile male generations for genetic vector control. Beyond genetic control, products of spermatogenesis genes can also be targeted by new mosquito-specific sterilizing compounds.
In parallel with these activities we are exploring possibilities of collaboration with colleagues at the Pirbright Institute to understand molecular mechanisms of vector-virus interactions.
 
Description We have identified a male-specific gene, named Yob, which is a master regulator of a pathway controlling sex determination in an emerging model organism, the mosquito Anopheles gambiae. Yob is located on the Y chromosome, and as such, is present exclusively in males. We have discovered that misregulation of Yob expression is sex-specific embryonic-lethal. Silencing of Yob expression kills males, whereas ectopic delivery of Yob mRNA into unsexed embryos kills females, leaving males unaffected. This unusual phenotypic effect suggests that Yob controls dosage compensation and that misregulation of Yob leads to a lethal destabilization of the dosage compensation machinery. Dosage compensation is a process, by which, in species with heterogametic males (possessing XY chromosomes) and homogametic females (XX karyotype), gene expression levels from a single male X chromosome are equalized with the expression levels from two X chromosomes in females. Molecular mechanisms to achieve this task vary in different organisms. Our analysis of global gene transcription in male and female Anopheles gambiae larvae and pupae shows that the male X chromosome is hypertranscribed in somatic tissues to the levels of female expression. However, in the testis transcription from the X chromosome is apparently not upregulated. Our preliminary analyses indicate that the elements of the dosage compensation machinery in Anopheles differ from those in Drosophila.
We have generated over 10 transgenic An. gambiae lines with perturbed Yob expression. In one of the lines with stable ectopic Yob expression in both sexes, all the females die during the embryo stage. In other lines we observe strong male bias, with the females surviving to the adult stage being strongly masculinized, i.e., possessing male-like head appendages (antennae and maxillary palps) and abnormally developed terminal segments of the abdomen; they do not feed on blood and die shortly after emergence, presumably due to dosage compensation issues. In other transgenic lines the Y chromosome-linked Yob is inactivated in certain somatic cells, and some males from these lines have strongly feminized terminalia.
In addition, we have analysed the function of selected An. gambiae genes involved in spermatogenesis. The RNA in situ hybridization to whole testis revealed that all the 16 genes analyzed are active in the pre-meiotic spermatocyte cells. However, stable knock-out of the subset of these genes through incorporation of the transgenic marker cassette to disrupt their coding sequence resulted in no discernible phenotypes. Thus, these genes must perform non-essential male reproductive functions, similar to many recently emerged genes in Drosophila.
Exploitation Route The data generated in this award served as preliminary information to secure further funding to explore in details the sex determination pathway in Anopheles mosquitoes. Generation of a transgenic line producing male only progenies provides a milestone platform to generate conditional female-lethal Anopheles lines for malaria control. In due course we plan to seek funding to perform a larger scale study to generate such transgenic strains with various conditional female-killing transgene constructs to identify strains with the highest male fitness necessary for field releases in mosquito control programs. Following semi-field trials, in collaboration with colleagues from endemic countries and from the International Atomic Energy Agency, we will perform open field test releases of irradiation-sterilized transgenic males, and if successful, larger-scale control operations. Although releases of non-sterile transgenic males would be more effective in suppression of local populations, irradiation sterilization programs are more acceptable to the public than open releases of transgenic strains. In addition to the above approaches, female-killing strains can be linked with other genetics-based control strategies, and, after obtaining the necessary approvals, used to introduce into and drive throughout the wild mosquito populations harm-reducing novel traits, such as inability to transmit pathogens.
An unequivocal demonstration that dosage compensation operates in mosquitoes opens new avenues of research into the components of the compensation machinery in this important group of vectors.
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology,Other

 
Description The discovery of a female-killing property of the primary sex determination gene is the first ever evidence that modulation of expression of a single gene can lead to female-specific embryonic lethality in mosquitoes. That opens an unprecedented opportunity for creation of mosquito transgenic strains, which under certain conditions would ectopically express the gene and kill all the females, allowing mass production of male-only populations for field releases in a range of genetic mosquito control programs. Only female mosquitoes bite. Therefore, such programs must incorporate male-only releases, because released females would contribute to pathogen transmission. As such, our discovery is expected to have an enormous impact on the control of mosquito-borne pathogens. We have created several proof-of-concept transgenic strains of Anopheles gambiae mosquitoes, in which we used expression of the primary sex determination gene as a tool to kill exclusively females. That work formed the basis to creation of transgenic mosquito strains with conditional female-killing tet-off system. The transgenic lines are yet to be tested for the activity of the system in vivo. If found to be working correctly, i.e., switching off the female killing in the presence of tetracycline, it would constitute a major step toward creating mosquito strains for releases in genetic control of mosquito-borne disease operations.
Sector Other
 
Description Components of the Medea gene drive system in a mosquito Aedes aegypti
Amount £1,209,348 (GBP)
Funding ID BB/S001964/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2018 
End 10/2021
 
Description Responsive mode
Amount £640,646 (GBP)
Funding ID BB/P019269/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description Components of the Medea gene drive system in a mosquito Aedes aegypti 
Organisation National Health Research Institutes (NHRI) Taiwan
Department Institute of Molecular and Genomic Medicine
Country Taiwan, Province of China 
Sector Charity/Non Profit 
PI Contribution Developed joined research project and lead successful application for funding
Collaborator Contribution Various lines of research, including construction of plasmids and development and characterisation of transgenic mosquito strains; and group discussions about results, conclusions and research progress.
Impact No impact yet.
Start Year 2018
 
Description Components of the Medea gene drive system in a mosquito Aedes aegypti 
Organisation National Institute of Genetics
Department NIG-FLY, Mishima (Drosophila RNAi resources)
Country Japan 
Sector Academic/University 
PI Contribution Developed joined research project and lead successful application for funding
Collaborator Contribution Various lines of research, including construction of plasmids and development and characterisation of transgenic mosquito strains; and group discussions about results, conclusions and research progress.
Impact No impact yet.
Start Year 2018
 
Description Reannotation of the Anopheles gambiae genome 
Organisation Biomathematics and Statistics Scotland (BioSS)
Country United Kingdom 
Sector Public 
PI Contribution Generated and provided RNA-seq data from the developmental transcriptome of the mosquito Anopheles gambiae Manually browsed the transcriptomic data in the genome context and provided coordinates for genome regions with not annotated genes or genes requiring annotation corrections. Generated and provided FAIRE-seq data from embryo samples of Anopheles gambiae to identify regulatory elements controlling early embryo development.
Collaborator Contribution Conducting reannotation of the Anopheles gambiae genome based on RNA-seq data from the developmental transcriptome. Mapping the FAIRE-seq data to the genome and linking the mapped data the RNA-seq data.
Impact No outputs yet
Start Year 2017
 
Description Reannotation of the Anopheles gambiae genome 
Organisation Centre for Genomic Regulation (CRG)
Country Spain 
Sector Academic/University 
PI Contribution Generated and provided RNA-seq data from the developmental transcriptome of the mosquito Anopheles gambiae Manually browsed the transcriptomic data in the genome context and provided coordinates for genome regions with not annotated genes or genes requiring annotation corrections. Generated and provided FAIRE-seq data from embryo samples of Anopheles gambiae to identify regulatory elements controlling early embryo development.
Collaborator Contribution Conducting reannotation of the Anopheles gambiae genome based on RNA-seq data from the developmental transcriptome. Mapping the FAIRE-seq data to the genome and linking the mapped data the RNA-seq data.
Impact No outputs yet
Start Year 2017
 
Description Reannotation of the Anopheles gambiae genome 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Generated and provided RNA-seq data from the developmental transcriptome of the mosquito Anopheles gambiae Manually browsed the transcriptomic data in the genome context and provided coordinates for genome regions with not annotated genes or genes requiring annotation corrections. Generated and provided FAIRE-seq data from embryo samples of Anopheles gambiae to identify regulatory elements controlling early embryo development.
Collaborator Contribution Conducting reannotation of the Anopheles gambiae genome based on RNA-seq data from the developmental transcriptome. Mapping the FAIRE-seq data to the genome and linking the mapped data the RNA-seq data.
Impact No outputs yet
Start Year 2017
 
Description Anna Azvolinsky, The Scientist, 30/06/2016 
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 Discussed implications for mosquito control of the discovery of the primary sex determining gene in the malaria mosquito Anopheles gambiae (published in Science, 353(6294):67-69, 2016).
Year(s) Of Engagement Activity 2016
URL https://www.the-scientist.com/?articles.view/articleNo/46458/title/-Maleness--Gene-Found-in-Malaria-...
 
Description EMBO Conference: Molecular and population biology of mosquitoes and other disease vectors: vector and disease control 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation on "Manipulation of the sex determination pathway in the African malaria mosquito Anopheles gambiae" given during the meeting held on 24 - 28 July 2017 in Kolymbari, Greece.
Year(s) Of Engagement Activity 2017
URL http://meetings.embo.org/event/17-vector-control
 
Description FAO/IAEA Coordinated Research Programme on methods of sex separation in mosquitoes; Research coordination meeting Bangkok 19-23 February 2018 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact FAO/IAEA Coordinated Research Programme on exploring genetic, molecular, mechanical, and behavioral methods of sex separation in mosquitoes; aimed at promoting specific areas of research, exchange of ideas and networking among the experts in the field.
Year(s) Of Engagement Activity 2013,2015,2016,2018
 
Description Holly Kernot, Veterinary Times, 13/06/2017 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Discussed potential of manipulating mosquito sex determination genes as tools for novel mosquito genetic control approaches.
Year(s) Of Engagement Activity 2017
URL https://www.vettimes.co.uk/news/study-to-be-transformational-in-mosquito-control-strategies/
 
Description Invited seminar at the National Health Research Institutes, Taiwan. 27 Feb 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A presentation describing research in my group on mosquito developmental biology and its potential uses for mosquito control; attended by primarily by members of the National Mosquito-Borne Diseases Control Research Center.
Year(s) Of Engagement Activity 2018
 
Description SMBE 2018 meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation entitled "Phylogenetically young genes and their function in Anopheles mosquitoes" given during the Society for Molecular Biology and Evolution 2018 meeting (8-12 July 2018) in Yokohama, Japan. Discussions held with potential collaborators on joined future research projects.
Year(s) Of Engagement Activity 2018
URL https://www.smbe2018.jp/