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Sex determination pathway in the malaria vector Anopheles gambiae

Final Report Summary - VECTRAP (Sex determination pathway in the malaria vector Anopheles gambiae)

Overview of Project Results
Anopheles gambiae mosquitoes are the major vectors of human malaria in Africa, posing an enormous burden on global health and economies. Every year 300–500 million people are infected by malaria and over a million people (mainly children) die as a consequence of P. falciparum infections. Disease control measures aimed at reducing mosquito populations have become less effective, largely due to the spread of insecticides resistance and associated logistical problems with their implementation. New vector control strategies that are affordable, easy to implement and sustainable are desperately needed. This global health problem has prompted an unprecedented effort aimed at generating new molecular tools through a better understanding of the biology and the genetics of anopheline mosquitoes.
In this scenario, the Vectrap project proposed as main objective to define the transcriptional programs selectively activated in male and female A. gambiae mosquitoes during embryogenesis, larval development and sex determination with the ultimate goal to elucidate key steps of the sex differentiation pathway and to identify the genes determining the mosquito ability to function as a vector for human malaria.
The importance of this objective stems from the fact that the only female mosquitoes act as disease vectors because unlike males that use nectar as energy source, they must feed on blood to generate their progeny. This implies that the genes controlling the differentiation of male and female phenotypes are crucial in determining the ability of female mosquitoes to transmit the disease.
A multidisciplinary approach involving high-throughput sequencing, bioinformatics, molecular biology and mosquito genetic manipulation has been utilized to achieve the intended objective. The next generation high-throughput sequencing technologies (RNA-seq) performed within the Vectrap project was instrumental to collect separately, in male and female mosquitoes, the complexity of the transcriptional program throughout mosquito development. The RNA-seq approach permitted the sampling of all classes of transcripts, including alternatively spliced mRNAs, microRNAs, and other non-coding RNAs with potential regulatory roles. The bioinformatics analysis performed to characterized the RNA-seq information permitted the quantitative analysis of transcripts during ontogeny in the two sexes, the discovery of genes that are differentially transcribed in coincidence with sexual differentiation. Specifically, it was possible to categorize 8.310 genes which passed quality control and had dN/dS and expression in two distinct expression datasets (7,518 on the autosomes and 792 on the X chromosome). The overall analysis revealed that female-biased genes evolve more rapidly than male ones. In addition, the development of dedicated software solutions and analysis workflows has been instrumental in identifying clusters of genes with similar sex-specific and temporal-specific expression with roles in fertility. Specifically, an innovative bioinformatic platform that enables to study transcriptional regulatory mechanisms as a function of chromosomal co-localization and expression together with a comparative platform permitted the identification of the regulatory regions active in controlling the expression of an important set of sex-specific reproductive genes (Accessory gland genes - Acps). On the wake of these first results, we brought forward the investigation to discover even more evidence of relationships between Acps and mosquito fertility and demonstrated that by silencing a large fraction of Acps in the males, females generated a reduced progeny when mated to these males.
Given theses results, we forged ahead with the study of the Acp reproductive genes, as differences in the Acp reproductive genes among anophelines, may provide insight into the origin and function of sex-related traits. To study reproductive genes we performed a reproduction Male accessory gland cluster comparative analysis: A dedicated analysis workflow was designed to perform the identification of putative orthologs of An. gambiae male accessory gland (MAG) genes localized to the 3R chromosome in 10 anopheline species (An. arabiensis, An. quadriannulatus, An. melas, An. merus, An. stephensi, An. funestus, An. minimus, An. dirus, An. atroparvus and An. albimanus). This analysis provided important information about Anopheles sex-specifc genes and their evolution. Mosquito reproductive biology evolves rapidly and presents a compelling target for vector control. This is exemplified by the An. gambiae male accessory gland protein (Acp) cluster on chromosome 3R: although in A. gambiae Acps cluster on chromosome 3R, this conservation is mostly lost outside the An. gambiae species complex, as revealed by the Vectrap bioinformatics analysis.
In this scenario, the Vectrap project in line with the need of the scientific community to significantly broaden the knowledge of mosquito biology to identify the genes determining the mosquito ability to function as a vector for human malaria provided invaluable and multifaceted results. Specifically, the Vectrap project addressed scientific and technical issues of great societal, economical and medical relevance.
a) Enhancing worldwide health and quality of life by fighting malaria
The Vectrap project provides a wealth of scientific and technical knowledge. The information acquired about the genetic aspects controlling the differentiation of male and female phenotypes may be useful for developing vector control solutions effective for fighting malaria. In this context, the achievements will include: i) deployment of an innovative analysis procedure to identify sets of genes likely to be concurrently involved in the same functional pathway; ii) identifying clusters of genes with similar sex-specific or temporal-specific expression or splicing patterns, with putative roles in ontogeny, sex determination, sex-specific development, gonadogenesis and gametogenesis; iii) annotation and characterization of those genes primarily responsible for altering mosquito fertility that will reveal potential new targets for inducing sterility; iv) high-throughput sequencing data of the A. gambiae transcriptome throughout a variety of conditions characterising the life span of the mosquito, drastically improving the current genome annotation.
b) Enhancing worldwide health and quality of life by targeting other insect species.
i) Health sector - While this project specifically targets the transcriptional programs selectively activated in male and female A. gambiae mosquitoes, many of its results are likely to be transferable to other insect species including Ae. Albopictus, Ae. Egypti and Culex, that act as vectors for several important parasitic and viral diseases and currently represent a real threat for Europe. ii) Agriculture sector - On a global scale, insects, plant pathogens and weed pests determine an overall 40% loss on food production on a yearly base. Insects alone are believed to cause a 10% to 25% destruction of gross national product in industrialized and developing countries. The possibility of transferring the results obtained with A. gambiae onto other species will pave the way to the development of population control strategies targeting those insect species that are responsible for the most significant economical losses in agriculture.
c) Fostering Academic Knowledge. This project appreciates the importance of sharing sequence data for the benefit of the overall scientific community. Proper bioinformatics resources will be used to annotate the sequence data that will also be archived through the 16 genone consortium in VectorBase, the major web-accessible database on invertebrate vectors of human pathogens. VectorBase data will allow the A. gambiae community to take full advantage of a wide array of consolidated tools and web applications for investigating the genome. In addition, the homology search tools made available in the database will provide benefit also to researchers working in other insects and interested in performing comparative genomics analysis.