Hossein Dehghan - Assistant Professor, Department Public Health, School of Health, Jiroft University of Medical Sciences, Jiroft, Iran.
Mohammad Ali Oshaghi - Professor, Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Seyed Hassan Moosa-Kazemi - Associate Professor, Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Bagher Yakhchali - Associate Professor, Department Industrial and of Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran
Hassan Vatandoost - Professor, Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Naseh Maleki-Ravasan - Assistant Professor, Malaria and Vector Research Group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

Background: Malaria is one of the most lethal infectious diseases in tropical and subtropical areas of the world. Following the mosquito insecticide resistance, Plasmodium drug-resistant and lack of an effective vaccine, paratransgenesis using symbiotic bacteria is nominated as new strategy to fight malaria.HasA). A group of mosquito with no bacteria was used as control. 2-5 days old female mosquitoes were fed with the bacteria [1×109cells/mL of 5% (wt/vol) fructose and red dye(1/50ml)] soaked in cotton pads for 24h. Then the sugar fed mosquitoes starved for 8h and allowed to feed for 30 min on P.berghei–infected mouse in dark condition at 17-20°C. The mosquitoes were maintained in 19±1°C and RH 80±5 and parasite infection was measured in different groups by counting the oocyst number 10 days post infection.Results: We reported an excessive (> 3,000 times) increase of E. cloacae in the midgut of mosquitoes 24h after blood meal. The wild and engineered bacterial strains could significantly (P< 0.0001) disrupt rodent malaria parasite P.berghei development in An. stephensi in comparison with the control group. The mean malaria inhibition of E. cloacae WT, E. cloacaeHasA, E. cloacaeS-HasA, and E. cloacaeGFP-D was measured 72%, 86%, 92.5% and 92.8% respectively.Conclusion: The synchrony of the bacterial proliferation and ookinete stage of Plasmodium in Anopheles midgut at 18-24 h after blood feeding provided an excellent chance for the secreted effector molecules to kill efficiently malaria parasite ookinetes, inhibiting oocyst formation. It is notable that E. cloacae wild type could significantly decrease P.berghei oocyst by intrinsic nature. This bacterium could be introduced and established in wild mosquitoes by attractive sugar bait to create a refractory mosquito population to plasmodium parasites. These finding provide the foundation for using paratransgenic An. stephensi by wild type or genetically modified E. cloacae as a powerful tool to fight malaria.Objectives: Here we evaluated two strains of engineered Enterobacter cloacae expressing different effector molecules to disrupt P.berghei ANKA 2.34 development in Anopheles stephensi midgut.Materials and Methods: Plasmodium berghei transmission-blocking assay was carried out by wild type(WT) and different engineered strains of E.cloacae dissolvens expressing GFP-defensin (GFP-D), GFP-HasA, and scorpine-HasA (S-

Paratransgenesis, Anopheles stephensi, Plasmodium berghei, Enterobacter cloacae, Transmission-Blocking Assays.