Microarray analysis for identification ofPlasmodium-refractoriness candidate genes in mosquitoes

Abstract

The identification and cloning of genes conferring mosquito refractoriness to the malaria parasite is critical for understanding malaria transmission mechanisms and holds great promise for developing novel approaches to malaria control. The mosquito midgut is the first major site of interaction between the parasite and the mosquito. Failure of the parasite to negotiate this environment can be a barrier for development and is likely the main cause of mosquito refractoriness. This paper reports a study on Aedes aegypti midgut expressed sequence tag (EST) identification and the determination of genes differentially expressed in mosquito populations susceptible and refractory to the avian malaria parasite Plasmodium gallinaceum. We sequenced a total of 1200 cDNA clones and obtained 1183 high-quality mosquito midgut ESTs that were computationally collapsed into 105 contigs and 251 singlets. All 1200 midgut cDNA clones, together with an additional 102 genetically or physically mapped Ae. aegypti clones, were spotted on single arrays with 12 replicates. Of those interrogated microarray elements, 28 (2.3%) were differentially expressed between the susceptible and refractory mosquito populations. Twenty-seven elements showed at least a two-fold increase in expression in the susceptible population level relative to the refractory population and one clone showed reduced expression. Sequence analysis of these differentially expressed genes revealed that 10 showed no significant similarity to any known genes, 6 clones had matches with unannotated genes of Anopheles gambiae, and 12 clones exhibited significant similarity to known genes. Real-time quantitative RT-PCR of selected clones confirmed the mRNA expression profiles from the microarray analysis.Key words: microarray, vector competence, real-time PCR, EST.