Malaria kills one African child every 30 seconds, according to the " Roll Back Malaria " program (WHO, UNDP, Unicef). Two billion human beings (40% of the world population) are exposed, and the number of clinical cases is estimated to be 300 million each year. There is still no vaccine against the disease and the causative parasite (Plasmodium) is growing more and more resistant to anti-malaria drugs. The fight against malaria hence rests heavily on the fight against the mosquito vector, Anopheles, which transmits the parasite to human beings. Unfortunately, this mosquito is becoming resistant to the available insecticides.

It is therefore crucial to understand the mosquito's biology and its interactions with the parasite in order to design new strategies to fight it.

To achieve this goal, the knowledge of the genome sequence and the study of its genes are a quick way to get data that will prove dramatically useful to scientists ; it may notably lead to new ways to limit the transmission of the parasite.

In the 1990's, several research groups had undertaken genetic studies of Anopheles, especially of its interactions withPlasmodium, which spends part of its life cycle in the insect's body. The identification of genes by classical means is however fastidious ; Anopheles specialists have therefore thought about sequencing large parts of the insect's genome, or even its whole genome. Thanks to progress generated by the Human Genome Program, such a project was no longer unrealistic.

France was the first to undertake large scale sequencing of the Anopheles genome. In 1998, Genoscope and the Unit of Insect Biochemistry and Molecular Biology at the Pasteur Institute have sequenced and analyzed the ends of 12 000 long genomic fragments, starting from a DNA library constituted by Frank Collins at Notre Dame University (USA). This first look at the Anopheles genome as a whole provided many chromosome markers, and led to the identification of new genes. But this piece of information was also useful in the perspective of sequencing the entire genome by assembling random pieces.

In March 2001 indeed, an international consortium for sequencing the Anopheles genome was formed. Genoscope participated in it by taking charge of part of the sequencing work. Most of the work was performed by the US company Celera Genomics, which received 9 million dollars in financing from the NIAID (National Institute of Allergy and Infectious Diseases).

The strategy adopted for sequencing the 280 millions base pairs (Mb) of the Anopheles genome was the " whole genome shotgun ", already used by Celera in 2000 for sequencing the genome of the fly Drosophila. It consists of reading the sequence of small random fragments (nearly 5 million, of which 500 000 had been sequenced at Genoscope) from the entire genome, representing many times its total length. The overlapping sequences are then assembled but, because of sampling, those assemblies (contigs) do not cover the whole genome and are therefore separated by "gaps", almost 20 000 in this case. Other sequence information, including Genoscope's preliminary data, were useful to order and orient the contigs in "scaffolds". In this way, Celera has covered most of the genome with a few large scaffolds.

The anchoring of scaffolds along the genome took advantage of a map established with "giant" chromosomes that exist in some tissues of the mosquito. The resulting draft of the genome has been freely available since March 2002. Genes were delimited and identified with informatics tools by Celera and Ensembl (joint project between the European Bioinformatics Institute and the Wellcome Trust in UK). However, this annotation work is far from being definitive. Moreover, the degree of polymorphism of the mosquito strain that was sequenced has caused difficulties during the assembly. Therefore, substantial modifications might occur when the finishing work will be undertaken. The Pasteur Institute and Genoscope will continue their collaboration in order to improve the quality and interpretation of the sequence data.

Barely three years ago, databases contained less than 10 complete genes from Anopheles. With the genomic sequence which is published today, scientists estimate that they now have 14,000 genes available. This wealth of information will allow them to better understand Anopheles biology. The identification of mosquito genes implicated in the transmission of the parasite, in the insect's resistance to insecticides, in its olfaction and immunity... should lead ultimately to the development of means to control malaria.

The analysis of the sequence has already led to better definition or reorientation of research projects. In the Unit of Insect Biochemistry and Molecular Biology at the Pasteur Institute, for example, scientists are interested in some proteins, the ABC carriers, that could be implicated in resistance to insecticides. Four of them were previously known, and we now have 50 of them ! And the analysis of the genomic sequence has already identified gene families that are worth working on.

Otherwise, comparisons between the Drosophila genomic sequence (obtained in 2000) and the mosquito sequence are extremely useful. For example, a mechanism which can block the parasite's development in the mosquito's body is also found in Drosophila, and a key gene involved in this mechanism has been found in this fly. Thanks to the Anopheles genomic sequence, the corresponding gene, a potential target for interrupting the parasite's development, was discovered very quickly in the mosquito by Pasteur's scientists. It would have taken much longer to localize it without this new data.

Many other potential applications will be accelerated. The mosquito's olfactory receptors, for example, are probably involved in the attraction of the female anopheline mosquito towards humans. A whole set of genes associated with olfaction has been discovered. This will greatly facilitate research on these receptors and will probably lead to the development of new mosquito repellants, or, on the contrary, of new attractive molecules. Otherwise, a better understanding of the metabolism involved in insecticide resistance would make it possible to use these products in a more ecological way.

It must also be said that the knowledge of the Anopheles genome will increase the number of potential target genes that could be modified by transgenesis in the mosquito. "I think that transgenic mosquitoes are a formidable tool in the laboratory, when kept in a confined area, because they will allow us to characterize genes of interest", says Paul Brey, head of the Unit of Insect Biochemistry and Molecular Biology at the Pasteur Institute. "However, transforming mosquitoes and liberating them in the field is at present, from my point of view, irresponsible. We do not know enough about the ecology of Anopheles in the field ; we do not have a single idea on how a population of transgenic mosquitoes would supplant natural populations. It would be risky to modify something without knowing if it is going to work in the desired direction. The applications that will come first, I believe, will concern a more rational use of insecticides and the development of new mosquito repellants."

To make best use of the data now available, the Pasteur Institute will launch a "large horizontal research program" dedicated to Anopheles : eleven teams from the parisian campus will participate in it, along with two institutes from the Pasteur Institutes and Associated Institutes International Network (Dakar and Madagascar). This program will be aimed in particular at the discovery and characterization of the genes implicated in the interactions between Anopheles and thePlasmodium falciparum parasite, whose genomic sequence is also published this week in the journal Nature.

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* The international Anopheles gambiae genome sequencing consortium includes Genoscope (Evry, France), the Pasteur Institute (Paris, France), Celera Genomics (USA), Notre-Dame University (USA), 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, USA), the Institute of Molecular Biology and Biotechnology (IMBB, Greece), under the auspices of the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (WHO/TDR, Geneva, Switzerland).

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Genoscope - French National Sequencing Center - is a large facility dedicated to genomic research. It puts its sequencing capacity at the research community's disposal within the framework of collaborative projects devoted to various organisms (microbes, plants, animals) of scientific, medical or economic interest. Involved in the Human Genome Project, the Genoscope has determined the sequence of the human chromosome 14. It also pursue its own projects, such as the sequencing of the fish Tetraodon nigroviridis, whose compact genome is a tool to annotate the human genome. Genoscope is based near Paris at Evry, in Essonne, France.

Press contact :
Yann Esnault - 33 (0)1 60 87 84 50 / yesnault@genoscope.cns.fr

The Pasteur Institute is a private, not-for-profit foundation. Since its creation, the Pasteur Institute has three major vocations : research in biology, principally aimed at infectious diseases, public health applications, and teaching. Since September 1999, the Pasteur Institute is being part of the "réseau national des génopoles" (genopoles national network) created by the French Ministry for Research and Technology. Several teams involved in the study of model or pathogen organisms have therefore begin to sequence the whole genome of these organisms and to study at large scale the structure and function of their genes.

Press contacts :
Nadine Peyrolo - 33 (0)1 45 68 81 47 / presse@pasteur.fr
Corinne Jamma - 33 (0)1 40 61 33 41 / presse@pasteur.fr

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