During the past 60 years, the amount of anthropogenic xenobiotics released into natural ecosystems has dramatically increased. Although the effect of these chemicals on human health is intensively studied, their impact on other organisms remains poorly understood. Because pollutants often accumulate in fresh-water bodies and sediments [1
], their impact on wetland fauna is of importance for these ecosystems. Among aquatic arthropods found in wetlands, mosquitoes are distributed worldwide and are often exposed to anthropogenic pollutants and insecticides during their aquatic larval stage. Indeed insecticides are often deliberately introduced into the mosquito habitat in the fight against the many human diseases they transmit (e.g. malaria, dengue fever, yellow fever and filariasis) [2
]. As a consequence mosquito control programs are now threatened by the selection of mosquito populations resistant to these chemical insecticides [3
]. Differential gene transcription in insecticide-resistant mosquitoes has been frequently used to identify genes putatively involved in inherited metabolic resistance mechanisms [4
]. For that purpose most approaches used cDNA microarrays and were often focused on genes encoding enzymes potentially involved in the bio-transformation of insecticides molecules [8
], although recent findings suggest that the differential expression of other transcripts may also contribute to insecticide tolerance [4
]. Less attention has been paid to the short term transcriptome response of insects to xenobiotics, though this may lead to the discovery of novel molecular mechanisms contributing to insecticide tolerance [11
]. We recently demonstrated that exposing mosquito larvae to low concentrations of pollutants for a few hours can increase their tolerance to chemical insecticides, possibly due to an alteration of the expression of detoxification enzymes [11
]. In this context, understanding cross responses of mosquitoes to insecticides and pollutants at the whole transcriptome level may ultimately lead to improvements in vector control strategies by optimizing insecticide treatments in polluted areas [7
]. Moreover, deciphering transcriptome response of mosquitoes to anthropogenic xenobiotics may identify genes involved in chemical stress response that were not detected by standard toxicological studies.
Today, quantitative transcriptomic methods are diversified and divided into two kind of technology: 'closed' and 'open' techniques depending on genome annotation constraints [14
]. In 'closed' technologies, gene expression microarrays are the standard method used for transcriptome analysis. However, this type of technology does not allow the characterization and analysis of new transcripts and suffers from various technical biases such as non-specific hybridization and insufficient signal for low expressed genes. In contrast, 'open' transcriptome analyses based on the sequencing of either ESTs or short cDNA tags, like Serial Analysis of Gene Expression (SAGE) [16
], LongSAGE [17
] and Massive Parallel Signature Sequencing (MPSS) [18
] can measure the transcript level of both known and unknown genes [19
]. The short cDNA tags obtained by LongSAGE or MPSS can directly be mapped to the genome sequence, allowing the identification of new transcripts [15
]. Because these sequencing techniques do not target a defined portion of cDNAs, these approaches are not optimized for the deep analysis of transcriptome variations [20
]. Recently, a combination of LongSAGE and Solexa sequencing technology, leading to the production and sequencing of millions of tags on a defined region of cDNAs, has been used to characterize mouse hypothalamus transcriptome [15
]. To our knowledge, this new method, called Digital Gene Expression Tag Profiling (DGETP) has never been used to compare whole transcriptome variations of a non-mammalian organism in different environmental conditions.
Here, we used the DGETP approach to perform a deep transcriptome analysis of larvae of the mosquito Aedes aegypti exposed to different anthropogenic xenobiotics. We examined the effect of sublethal doses of three pollutants likely to be found in wetlands (the herbicide atrazine, the polycyclic aromatic hydrocarbon fluoranthene and the heavy metal copper) and three chemical insecticides used for mosquito control (the pyrethroid permethrin, the neonicotinoid imidacloprid and the carbamate propoxur). This approach was suitable for investigating deep transcriptome variations in mosquitoes and identified several loci with high transcription signal not previously identified in mosquito genome. At the biological level, the transcript levels of many genes were affected by xenobiotic exposure. Several genes and protein families responding to individual or multiple xenobiotics were identified, unraveling the complexity of xenobiotic-response in mosquitoes and identifying genes potentially involved in insecticide tolerance or biological interactions between insecticides and pollutants.