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Background: Malaria still remains a serious health burden in developing countries, causing more than 1 million deaths annually. Given the lack of an effective vaccine against its major etiological agent, Plasmodium falciparum, and the growing resistance of this parasite to the currently available drugs repertoire and of Anopheles mosquitoes to insecticides, the development of innovative control measures is an imperative to reduce malaria transmission. Paratransgenesis, the modification of symbiotic organisms to deliver anti-pathogen effector molecules, represents a novel strategy against Plasmodium development in mosquito vectors, showing the potential to reduce parasite development. However, the field application of laboratory-based evidence of paratransgenesis imposes the use of more realistic confined semi-field environments. Methods: Large cages were used to evaluate the ability of bacteria of the genus Asaia expressing green fluorescent protein (Asaiagfp), to diffuse in Anopheles stephensi and Anopheles gambiae target mosquito populations. Asaiagfp was introduced in large cages through the release of paratransgenic males or by sugar feeding stations. Recombinant bacteria transmission was directly detected by fluorescent microscopy, and further assessed by molecular analysis. Results: Here we show the first known trial in semi-field condition on paratransgenic anophelines. Modified bacteria were able to spread at high rate in different populations of An. stephensi and An. gambiae, dominant malaria vectors, exploring horizontal ways and successfully colonising mosquito midguts. Moreover, in An. gambiae, vertical and trans-stadial diffusion mechanisms were demonstrated. Conclusions: Our results demonstrate the considerable ability of modified Asaia to colonise different populations of malaria vectors, including pecies where its association is not primary, in large environments. The data support the potential to employ transgenic Asaia as a tool for malaria control, disclosing promising perspective for its field application with suitable effector molecules. [ABSTRACT FROM AUTHOR]