Identification of bacteria associated with malaria mosquitoes - Their characterisation and potential use

Abstract: The use of transformed bacteria to stop or kill disease-causing agents in the gut of vector insects is called paratransgenics. Two of the major steps in creating a paratransgenic Anopheles mosquito, unable to spread the Plasmodium parasites that cause malaria, are to find a bacterium suitable for the purpose and a way to introduce the transformed bacterium into mosquitoes in the field. In this project, bacteria associated with malaria mosquitoes have been identified by phylogenetic analysis of their 16S rRNA genes. First, the midgut flora of field-caught Anopheles mosquitoes was examined using two pathways, one culture dependent and one culture independent. Second, six bacterial species from an An. gambiae laboratory colony, and third, ten isolates from Anopheles oviposition sites have been identified. Altogether, 32 bacterial species, representing 16 families, seven classes and four phyla were identified. Interestingly, several of them are related to bacteria known to be symbionts in other insects. Two possible ways of introducing bacteria into mosquitoes in the field in a paratransgenic approach were investigated in a laboratory setting. It was shown that sugar solutions with or without bacteria are equally attractive to An. gambiae mosquitoes and that the mosquitoes were able to take up bacteria from the water they emerged from. These results show that it may be possible to use sugar-baits and oviposition sites for distribution of genetically modified bacteria in the field. To facilitate the distribution of the modified bacteria mosquito attractants should be used. We investigated whether the bacterial isolates identified in this project produce attractants affecting mosquito sugar-feeding or oviposition site selection. While no responses were observed from the mosquitoes towards bacteria-containing sugar solutions, seven of the 19 isolates examined mediated positive oviposition responses. In total, 13 putative oviposition attractants were identified among the volatiles emitted by the attractive bacteria.