Mechanisms of sex determination in Anopheles and their implementation to control mosquito vectors

Lead Research Organisation: The Pirbright Institute
Department Name: Vector Molecular Biology

Abstract

The molecular developmental processes that determine gender in animals are astonishingly variable. Because genes governing these processes often undergo rapid changes, information on the components of the sex determination pathway gathered in one species may provide little or no clue to facilitate identification of the sex determination genes in other, more distantly related, species. In insects, the pathway has been characterised in detail in the fruit fly Drosophila melanogaster; however, despite efforts, it remains unexplored or poorly understood in other insect groups, including mosquitoes. Recently, we have identified a gene (named Yob), which in the malaria mosquito, Anopheles gambiae, is located on the male-specific Y chromosome and controls the development of an individual into a male. Surprisingly, Yob kills female embryos, if expressed (activated) during early steps of female development. The mechanisms through which Yob regulates male development and kills females remain unknown. To fill these knowledge gaps, we propose exploration of an extensive, high-throughput sequence data of genes active during the development of A. gambiae. The data, already generated by our group and collected separately for males and females at a number of time points, starting from early embryos to adults, will be filtered to identify sex-specifically expressed genes that may be involved in the mentioned above processes. In a parallel approach, we will perform a molecular study of genetically modified mosquito strains, with Yob either inactivated in males, or activated in females, to detect genes with perturbed expression, indicative of their direct or indirect interaction with Yob. Such transgenic strains were recently generated by our group and are available for this study. The role of the identified genes in sexual development of Anopheles will be tested by a number of experimental approaches. We will also identify DNA regulatory regions in the A. gambiae genome necessary to activate gene expression in early embryos, and evaluate their function in transgenic mosquitoes. Our proposed research will significantly contribute to the understanding of insect sex determination pathways, and is expected to have a great impact on novel strategies of mosquito control. The sex determination genes, when manipulated in transgenic technology, could be used to eliminate females and produce male-only generations by causing female lethality or, potentially, sex reversal of genetic females into males. In a broader context, the outputs of this study will facilitate identification of sex determination genes in other insect pest species, including mosquito vectors of arboviruses, and will stimulate new avenues of research on genetic control of these insect groups.

Technical Summary

An almost universal occurrence of two genders and sexual reproduction in most animal groups implies existence of a common underlying mechanism of sex determination. Yet, this fundamental developmental process is regulated in an astonishing variety of ways. Because of their rapid evolution, the sex determination pathway genes are often difficult to identify. Therefore, despite being well characterized in Drosophila, the pathway remains unexplored or poorly understood in other insects, including lower dipterans, such as mosquitoes. Recently, we have identified the primary sex determination gene (called Yob) in the mosquito Anopheles gambiae by comparing male and female embryo transcriptomes. Yob is Y chromosome-linked and confers maleness via an unknown mechanism. Intriguingly, ectopic expression of Yob during early development kills female embryos, likely through inappropriate activation of dosage compensation. Neither the components of the dosage compensation complex in Anopheles are known, nor how the sex determination and dosage compensation are interconnected. The proposed study aims at exploring these processes to fill the existing knowledge gaps. Genes mediating the maleness signal from Yob will be targeted by comparing male and female developmental transcriptomes and by focusing on genes with the sex-specific transcripts or sex-specifically spliced isoforms. In parallel, we will study the transcriptomes and proteomes of the wild-type and transgenic mosquito strains with perturbed Yob transcription to pinpoint the molecules affected by Yob mis-expression. The role of the candidate genes in sex determination and dosage compensation will be validated by gene silencing or overexpression in cell lines and in the Anopheles embryos. In addition, we will use FAIRE-seq to conduct genome-wide survey of early zygotic promoters and will test the activity of the selected promoters in vivo by creating Anopheles strains with conditional Yob-driven female lethality.

Planned Impact

This study will provide data and resources that will have a direct influence on translational research on vector and pest control. In particular, information on the sex determination pathway genes and early embryonic promoters is crucial to the advance of novel genetic control approaches. Most genetic control strategies targeting mosquitoes must incorporate male-only releases, because released females would contribute to pathogen transmission (males do not bite). No methods to sex mosquitoes on a sufficiently large scale exist; thus, transgenic strains conditionally producing male-only generations will be necessary for releases in control operations. Identification of yet unknown elements of the sex determination pathway will significantly increase flexibility in the design of transgenic systems aimed at elimination of mosquito females. From the economic standpoint it is of prime importance that females are removed at an early stage of development, and doing so during the embryonic stage will drastically reduce costs of production of high quality males. Transgenic Anopheles strains with conditional female-lethality developed in this study could potentially be used in pilot field trials of sterile insect technique (SIT) in certain settings.
Genetic control is highly species-specific and does not entail the spread of noxious chemicals in the environment. Therefore, it is a very attractive strategy to control harmful insects. We expect that better understanding of the sex determination pathway in Anopheles will greatly increase the power of comparative genomics methods to identify sex determination genes in other insects, eventually enabling the development of transgenic sexing strains and novel genetic approaches to the control of mosquito vectors of arboviruses and of insect pests. From this perspective, our study will have a major impact on both mosquito and pest research and control. In result, it will lead to an improved human and animal health and wellbeing, and increased agricultural productivity with minimal environmental impact. Therefore, in the long term the ultimate beneficiaries of the study will be hundreds of millions of people who, because of the harm inflicted by various insect pests, suffer from bites, vector-borne diseases, poverty and hunger. Our results will be of interest to both general public and to the policy makers, especially of the international bodies, such as the World Health Organization, the Food and Agriculture Organisation of the United Nations (FAO) and the International Atomic Energy Agency (IAEA). FAO and IAEA play a leading role in the development and implementation of the genetic sexing-based SIT technology to control mosquitoes and insect pests. Since the outputs of this study are expected to directly lead to the development of commercially exploitable products, our results will also be of interest to industrial partners. The results of our research will be disseminated to the broad audience using various means. Important breakthroughs will be channelled through media: local, national and international, where appropriate, by the communications team at the Pirbright Institute. In addition, significant achievements will be publicised on the Institute's website and social media accounts. Furthermore, the web pages of the applicants, that are accessible to the general public and dedicated to their current research, will be regularly updated to reflect progress of work. We will also use our contacts with colleagues from IAEA to ensure translation of our findings into insect control technology. These activities will be regulated by formal collaboration agreements prepared with the assistance of a legal team at the Institute to protect intellectual property of the outputs of our study.

Publications

10 25 50
 
Description We have identified and isolated yet unknown regulatory elements (promoters) driving gene expression in embryos of the African malaria mosquito, Anopheles gambiae. The function of the promoters has been validated in transient assays in A. gambiae embryos. One of the promoters has been used in a transgenic plasmid construct to activate in both sexes the sex determination gene Yob conferring maleness. Using that construct, or its derivatives, we generated a number of genetically modified strain of A. gambiae, which produces male-only progeny, because females die during the embryo stage. In addition, we have generated important new resources: over 10 A. gambiae docking lines - genetically modified strains, which allow precise integration of new transgenic constructs into well characterised locations within the genome. We have also made progress with identification of new genes or candidates involved in the sex determination pathway in Anopheles. One gene located downstream of Yob has been validated as the pathway's element vital for female development. We have generated several transgenic A. gambiae lines, in which transcripts of that gene are stably depleted, leading either to female lethality or profound developmental defects preventing them from blood feeding. Three further sex determination candidate genes are currently under study. Overall, these results shed new light on the sex determination pathway in mosquitoes and represent an important step towards creation of novel genetic mosquito control tools.
Exploitation Route The data gathered, in particular identification of a gene that is vital for female development and the information about A. gambiae transgenic strains producing male-only progenies is an important step in generation of genetic tools for vector-based malaria control.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Other

 
Title A male-specific cell line from a mosquito Anopheles gambiae 
Description A new male-specific cell line from a mosquito Anopheles gambiae has been isolated from sexed neonate larvae. To our knowledge this is the only cell line derived exclusively from male mosquitoes. 
Type Of Material Cell line 
Year Produced 2019 
Provided To Others? No  
Impact The cell line will greatly facilitate studies on components of the sex determination pathway and of dosage compensation machinery in mosquitoes. There are only several cell lines derived from Anopheles gambiae available and these are female-like. Over-expression of genes involved in male sex determination or dosage compensation is detrimental to female cells, hence use of male-derived cells is necessary for such studies. The cell line will become available upon acceptance of a manuscript describing characterization of that cell line. 
 
Description Linking sex determination and dosage compensation in a mosquito Anopheles gambiae 
Organisation University of Zurich
Department Institute of Molecular Life Sciences
Country Switzerland 
Sector Academic/University 
PI Contribution Developed a new research program.
Collaborator Contribution Provided intellectual input, contributed to drafting a joined grant proposal.
Impact No output yet.
Start Year 2019
 
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 1st Research Coordination Meeting (RCM) of the Coordinated Research Project (CRP) on "Generic approach for the development of genetic sexing strains for SIT applications" Vienna, 7-11 October 2019 
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 Project on exploring genetic and molecular methods of sex separation in insect pests; aimed at promoting specific areas of research, exchange of ideas and networking among the experts in the field.
Year(s) Of Engagement Activity 2019
 
Description EMBO Workshop: 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 "Sex determination pathway in Anopheles gambiae as a target for mosquito control" given during the meeting held on 22 - 26 July 2019 in Kolymbari, Greece. Following a presentation a request was made to give an informal seminar at the Department of Life Sciences, Imperial College, London.
Year(s) Of Engagement Activity 2019
URL https://meetings.embo.org/event/19-mosquitoes
 
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 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 Participation in a meeting regarding sex determination, Duesseldorf, Germany 
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 "Sex determination pathway in Anopheles gambiae as a target for mosquito control" during the Nothiger meeting on sex determination in insects, Duesseldorf, 11-13 March 2019.
Year(s) Of Engagement Activity 2019
 
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/
 
Description School visit (Farnborough) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Gave a presentation to a group of students about research performed at the Pirbright Institute and discussed various aspects of scientists' work.
Year(s) Of Engagement Activity 2018
 
Description School visit (Tillingbourne) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Gave a presentation to a group of students about role of insects in the environment; guided students in collection of insects and other invertebrates, and identification of collected material.
Year(s) Of Engagement Activity 2019