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

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.

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.

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.