Positive selection drives accelerated evolution of mosquito salivary genes associated with blood feeding.

INTRODUCTION : The saliva of bloodsucking animals carries dozens to hundreds of proteins whose main role is to counteract their hosts’ haemostasis, inflammation and immunity. Previous studies on both anopheline and culicine mosquitoes highlighted the high rate of interspecific diversity of salivary (S) proteins as compared to housekeeping (H) ones. In An. gambiae-An. stephensi [Ae. aegypti-Ae. albopictus] S proteins are 62.4% [71.5%] identical whereas H proteins share 93.1% [94.0%] amino acid residues. This pattern may be explained by relaxed selection or random drift. Alternatively, may be the result of positive selection driven by the intense pressure of the host immune system on proteins/genes that are essential for blood feeding and, therefore, strongly affect mosquito reproduction/fitness. In such a scenario, host antibody response to salivary proteins may favour the selection of protein variants with conserved biological functions and different antigenic properties. MATERIALS AND METHODS : To test our working hypothesis we looked for selection signatures in five salivary genes involved in blood feeding (Apy, D7r2, gSG6, gSG7, gVAG). Genes were amplified from individual An. gambiae s.s. mosquitoes (n=63-81) collected in October 2008 in the village of Soumousso (Bobo-Dioulasso, Burkina Faso). For each gene equimolar amounts of the amplified products were mixed and used to construct plasmid libraries. Recombinant clones (192/library) were randomly picked and sequenced. The rpS7 ribosomal protein gene was used as quality control. Sequences were aligned by the cap3 assembler, edited by BioEdit and analyzed using DnaSP, MEGA and HyPhy. RESULTS : Overall, 873 SNPs (395 coding, 478 noncoding) were identified, which corresponds to ~11.1 SNPs/kb; in comparison 9.2 and 8.9 SNPs/kb were found in a previous study for 72 immune and 37 non-immune An. gambiae genes. gSG6 was under strong purifying selection, whereas gSG7 appeared to evolve at a very fast rate. For all genes analyzed dN/dS<1 which indicates overall negative selection acting on the salivary genes. However, when positive selection was tested on individual codons by the HyPhy program using both the FEL (fixed-effects likelihood) and the MEME (mixed-effects model of evolution) models, nearly all salivary genes showed one or more codons with significant signatures of positive selection. CONCLUSIONS : According to the neutral theory most of the variability/change within a species is caused by random drift of selectively neutral mutants (non affecting fitness). Against this pervasive random drift selection may occur, with negative selection removing deleterious mutations and positive (Darwinian) selection promoting beneficial ones. Positive selection is rare and most of the selection signatures deviating from neutral expectations are found for negative selection. Exceptionally, cases for positive selection are found in genes associated with immunity, mate- and self-recognition, or with genes associated with virulence in pathogen recognition. Mosquito salivary genes associated with blood-feeding are shown here to be under strong positive selection, which explains their fast rate of divergence. We believe that our study provides evidence for a new class of positively selected genes, those coding for salivary proteins involved in bloodsucking by arthropods, and, specifically, the salivary proteins of the malaria vector An. gambiae.