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The mosquito Anopheles gambiae is the principal vector of malaria in Africa. According to the latest WHO statistics, this parasitic disease infects from 300 to 500 million persons per year in the world, and kills more than a million and a half each year, mainly African children. Together with AIDS, malaria is one of the principal causes of mortality in the populations of Africa, Southeast Asia and Latin America ; it contributes in large part to the continued impoverishment of these populations.
The parasites of the Plasmodium genus, which are the cause of the disease, are transmitted to humans by the bite of an infected female mosquito. One attempts to limit transmission by the use of mosquito nets and anopheline mosquito eradication campaigns. But the mosquito has developed resistance to insecticides and in some areas the parasite has developed resistance to anti-malarial drugs.
A better knowledge of the Anopheles mosquito will lead to better ways to control it, and also to target a new stage in the life cycle of Plasmodium. Of the relationships between the three actors in the disease -the parasite, its human host and its dipteran vector -, the most well-known to date is the interaction between Plasmodium and the infected human ; however this research has not yet led to decisive advances in the development of a vaccine. On the other hand, there has been little research on the interactions between the parasite and the insect. These studies might reveal new strategies for inhibiting the development of the parasite in the mosquito and its transmission to humans from mosquitoes. Finally, the interaction between the mosquito and humans - the attraction of the female mosquito to humans - depends on olfactory and taste receptors for which it will be important to identify the genes : this could lead to the development of new repellants, or attractive molecules to be used in odor traps.
The importance of the Anopheles genome project.
The identification of genes implicated in immunity of the mosquito to the parasites which cause malaria, in attraction to humans or in resistance to insecticides represents a tedious job if classical genetic methods are used. Thus, with the progress in sequencing of higher organisms, the ambitious project of attacking the complete genome of Anopheles was born. Beginning as early as 1998, Genoscope and the Insect Biochemistry and Molecular Biology Unit of the Pasteur Institute embarked on a large-scale sequencing program. Following this initiative, an international consortium was formed at the Pasteur Institute in April 2001 with the goal of the complete sequencing of the Anopheles genome ; the bulk of the sequencing work was to be undertaken by the American company Celera Genomics and Genoscope.
The source of genomic DNA the consortium selected to sequence is the PEST strain. This strain of Anopheles was produced by crossing mosquitoes collected in East Africa with a laboratory strain which carried a mutation - pink eye - which is useful for detecting contaminant mosquitoes. Frank Collins’ group at the University of Notre Dame (USA) had already used this strain for the preparation of a library of large genomic fragments cloned in bacterial artificial chromosomes (BACs), which was used at the beginning of the project. Using a highly polymorphic species from the field, rather than a pure strain as in other sequencing projects, constituted both a novelty and a problem. This situation will probably occur more and more often as genomics seeks to explore ecologic diversity.
Today the consortium has achieved its first objectives : a “working draft” of the sequence of the Anopheles genome has been freely accessible since March 2002, and the article which describes this draft appeared in Science magazine on October 4, 2002. Because of the sequencing strategy (see Sequencing project), the draft consists of about 19,000 contigs (assemblies of overlapping reads), which are themselves connected to about 9,000 “scaffolds”. Thus it is not yet a “finished” sequence, which limits the search for genes. Furthermore, the polymorphism of the PEST strain, due to its origin, has caused problems and uncertainties in the assembly. Genoscope and the Pasteur Institute are continuing their collaboration in an effort to improve the quality and interpretation of the sequencing data.
The draft sequence of the Anopheles genome
Despite its imperfections, the available sequence already constitutes an extraordinary tool for the community of scientists working on the mosquito, and more generally, on malaria. The annotation (delimitation and characterization of the genes) has been undertaken by Celera and the European bioinformatics group Ensembl, by combining homology searches and ab initio prediction using algorithms. The availability of the genomic sequence of another dipteran insect, Drosophila, which has been the target of genetic studies for almost a hundred years, is also an advantage. Around 14,000 genes have been defined so far and delivered to satisfy the curiosity of mosquito specialists since March 2002, whereas in 1999 only a dozen were known ! These automatic annotations must still be verified “manually”, and functional genomics and transgenesis projects will have to be undertaken to experimentally establish the role of these genes.
Among the first results of the exploration of the Anopheles genome is the discovery of about 20 new members of a family of olfactory receptors absent in Drosophila. This could be the point of departure for an understanding of the signals which guide the female mosquito to humans, and the conception of attractant or repellent molecules. Furthermore, the identification of taste receptors could elucidate the mechanism by which the mosquito identifies its host. Another difference from Drosophila is the higher number of proteases implicated in the innate immune response in insects, which might reflect the more exposed life-style of Anopheles. These genes, as well as others, could be involved in the mosquito’s response to infection with Plasmodium, and thus constitute targets for blocking the development of the parasite in the insect. Also of interest are the ABC transporters ; these are proteins which seem to be implicated in resistance to insecticides, and Pasteur’s scientists are already working on these molecules. Only four were known before the sequencing project - now there are about 50 of them. Genes which are activated or repressed during the blood meal of the mosquito have also been studied. The development of DNA chips from the sequences of Anopheles genes will make it possible to study their expression more precisely.
Research at the Pasteur Institute illustrates the importance of comparative genomics for the study of Anopheles genes. A mechanism known as melanisation, which blocks the development of the parasite in Anopheles, also operates in Drosophila, and a gene which regulates it is known in this fly. When the sequence of Anopheles became available, a homologous gene was rapidly discovered in the mosquito. Conversely, the genome of Anopheles could serve to interpret those from other species of mosquitoes which are vectors of parasitic diseases, such as Aedes aegypti, which is the vector for dengue, and for which sequencing has just begun.
This draft, combined with the sequences of the Plasmodium falciparum genome and the human genome, creates a unique situation in the field of infectious diseases, in which genomic information about the parasite, its vector and its host is now available. Numerous research avenues are now open, in which new research groups may become implicated, which has led the CNRS to launch a grant programme “Post-sequençage anophèle” in April 2002 with the support of the Pal+ Programme of the Ministry of Research. Priority targets include the phylogeny of Anopheles and analysis of polymorphisms in natural populations, which could contribute to the success of various anti-mosquito strategies in the field. Additionally, the Pasteur Institute has launched a “grand programme horizontal de recherche” centered on Anopheles, with the participation of 11 groups from the Paris campus as well as Pasteur Institutes in Dakar and Madagascar.
Of the many results of the Anopheles genome sequencing, it is difficult to predict which ones will be the most useful in the fight against this mosquito and against Plasmodium, but it seems that we now have sufficient information to frustrate the fantastic capacities of adaptation of these two organisms.
The International Consortium for the Sequencing of Anopheles genome includes Genoscope, the Pasteur Institute (Paris), Celera Genomics (United States), the University of Notre-Dame (United States), Ensembl (joint project between the Wellcome Trust and the European Bioinformatics Institute (United Kingdom)), The European Molecular Biology Laboratory (EMBL, Germany), The Institute for Genomic Research (TIGR United States), the Institute of Molecular Biology and Biotechnology (IMBB, Greece), under the auspices of the United Nations Development Program/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases (WHO/TDR, Geneva, Switzerland).