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Divergent selection based on aquatic larval ecology is a likely factor in the recent isolation of two broadly sympatric and morphologically identical African mosquito species, the malaria vectors Anopheles gambiae and An.coluzzii. Population-based genome scans have revealed numerous candidate regions of recent positive selection, but have provided few clues as to the genetic mechanisms underlying behavioural and physiological divergence between the two species, phenotypes which themselves remain obscure. To uncover possible genetic mechanisms, we compared global transcriptional profiles of natural and experimental populations using gene-based microarrays. Larvae were sampled as second and fourth instars from natural populations in and around the city of Yaounde, capital of Cameroon, where the two species segregate along a gradient of urbanization. Functional enrichment analysis of differentially expressed genes revealed that An.coluzzii - the species that breeds in more stable, biotically complex and potentially polluted urban water bodies - overexpresses genes implicated in detoxification and immunity relative to An.gambiae, which breeds in more ephemeral and relatively depauperate pools and puddles in suburbs and rural areas. Moreover, our data suggest that such overexpression by An.coluzzii is not a transient result of induction by xenobiotics in the larval habitat, but an inherent and presumably adaptive response to repeatedly encountered environmental stressors. Finally, we find no significant overlap between the differentially expressed loci and previously identified genomic regions of recent positive selection, suggesting that transcriptome divergence is regulated by trans-acting factors rather than cis-acting elements.

A speciation process is ongoing in the primary vector of malaria in Africa, Anopheles gambiae. Assortatively mating incipient species known as the M and S forms differentially exploit larval breeding sites associated with different ecological settings. However, some ongoing gene flow between M and S limits significant genomic differentiation mainly to small centromere-proximal regions on chromosomes X and 2L, termed 'speciation islands' with the expectation that they contain the genes responsible for reproductive isolation. As the speciation islands exhibit reduced recombination and low polymorphism, more detailed genetic analysis using fine-scale mapping is impractical. We measured global gene expression differences between M and S using oligonucleotide microarrays, with the goal of identifying candidate genes that could be involved in this ongoing speciation process. Gene expression profiles were examined in two independent colonies of both forms at each of three developmental periods of interest: fourth instar larvae, virgin females, and gravid females. Patterns were validated on a subset of genes using quantitative real-time reverse transcription polymerase chain reaction of RNA samples from laboratory colonies and wild mosquitoes collected from Cameroon and Burkina Faso. Considered across all three developmental periods, differentially expressed genes represented approximately 1-2% of all expressed genes. Although disproportionately represented in the X speciation island, the vast majority of genes were located outside any speciation island. Compared to samples from the other developmental periods, virgin females were characterized by more than twice as many differentially expressed genes, most notably those implicated in olfaction and potentially, mate recognition.