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.
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.
Organisations
Publications
Krzywinska E
(2022)
Establishment and a comparative transcriptomic analysis of a male-specific cell line from the African malaria mosquito Anopheles gambiae.
in Scientific reports
Krzywinska E
(2023)
A novel factor modulating X chromosome dosage compensation in Anopheles.
in Current biology : CB
Krzywinska E
(2021)
femaleless Controls Sex Determination and Dosage Compensation Pathways in Females of Anopheles Mosquitoes.
in Current biology : CB
Krzywinska E
(2018)
Effects of stable ectopic expression of the primary sex determination gene Yob in the mosquito Anopheles gambiae.
in Parasites & vectors
Description | We have identified, in the African malaria mosquito Anopheles gambiae, a new gene controlling sex determination and dosage compensation pathways. Named femaleless (fle), the gene represents a novel and the only known link between sex determination and dosage compensation pathways in non-drosophilid insects. fle is essential for normal female development (through controlling dsx, the terminal gene of the sex determination pathway, splicing into female form) and survival, and its function is conserved in various (likely all) vectors of human malaria. dsx splicing in Drosophila females depends on interaction of tra and tra2 genes. Evidence gathered during our study suggests that fle, which is only remotely similar to tra2, may not need an interacting tra-like partner for its function. We have identified three other genes of interest, with sex-specific splicing in all developmental stages. One of these genes, named 007, was characterised in detail. 007 is sex-specifically spliced in all developmental stages of A. gambiae and produces transcripts encoding longer male-specific and shorter female-specific 007 protein isoforms. 007 splicing is under a direct control of the fle gene, which in females causes 007 intron retention and a concomitant production of a shorter protein. Transient RNAi knockdown experiments in the early embryos targeting a 007 sequence common to both sexes yielded no sex bias in surviving individuals, indicating that the gene does not belong to the sex determination pathway. This notion was also supported by a stable 007 knockdown in a transgenic line, phenotype of which included slow development and a smaller size of both males and females as compared to the wild-type mosquitoes, but no evident sex bias. To further test 007 function, we generated two deletion mutant lines, in which excision of different fragments of the 007 gene led to deletions in the male-specific protein isoform. In both mutant lines the male development was slower than in the wild-type males, suggesting that dosage compensation (DC) is affected. To establish if this is the case, we generated RNA-seq data for male pupae of the deletion mutant lines and compared it to the RNA-seq data from the wild-type pupae. Unlike for the autosomes, expression from the X chromosome was significantly lower in the mutant lines, which provides unequivocal evidence that 007 is a component of the DC in Anopheles. It is a highly significant finding; Drosophila is the only insect in which DC mechanism has been identified at the molecular level, and the homologous mosquito molecules are not involved in regulation of DC. Thus, our discovery marks the Anopheles 007 as an element of a novel complex regulating expression of the X chromosome in male insects. Using a combination of RNA-seq data and FAIRE-seq reads (corresponding to open chromatin regions) in early embryos, we have identified numerous putative promoters/enhancers active during early development. We have isolated selected putative promoters and validated their activity in A. gambiae embryos through transient assays. One of the promoters has been used in a transgenic plasmid construct to activate, in both sexes, the sex determination gene Yob, which confers maleness. Using that construct, or its derivatives, we generated genetically modified A. gambiae strains, that produce exclusively male progeny, because females are killed in the embryo stage. Moreover, we generated transgenic strains with stable knockdown of fle transcripts, which in females resulted in either lethality or profound developmental defects. We also generated a number of transgenic lines with similar deleterious effects on females caused by ectopic expression of Yob ¬¬ through the tet-off system. Fortuitously, microinjection of embryos with the tet-off construct resulted in the recovery of five independent docking lines with the Y chromosome-linked transgene. Several of the tet-off Yob lines were tested for conditional lethality of the construct through the addition of doxycycline to blood used for feeding females, sugar solution used for feeding by both sexes, and to the water used for egg laying and larval development, to block Yob expression. However, the deleterious effects persisted despite supplying different concentrations of antibiotic, which indicates the need for improvements of the existing system. Finally, we have isolated , established, and molecularly characterised the first male-specific cell line from A. gambiae to facilitate studies on components of the sex determination pathway and of dosage compensation machinery in mosquitoes. |
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. Sex-specific splicing of the 007 gene can be also exploited in the transgenic constructs aimed at eliminating females. The Y chromosome-linked transgenic docking lines are, in general only rarely isolated and, thus, represent an important resource, for example for creating Y-linked X-shredding gene drives. Recently we shared the Y-linked docking lines with colleagues from the Hebrew University of Jerusalem upon their request. |
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 | Y chromosome-linked X-shredding gene drives |
Organisation | Hebrew University of Jerusalem |
Country | Israel |
Sector | Academic/University |
PI Contribution | We shared with the partner Y chromosome-linked docking lines generated in our lab. |
Collaborator Contribution | Provided some plasmid constructs. |
Impact | No outputs yet. |
Start Year | 2021 |
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 |