Dynamics of engineered Enterobacter cloacae GFP-D in Anopheles stephensi, its breeding place and fructose solution Under Laboratory and semi-field Condition

Background: Enterobacter cloacae is a known Anopheles gut microflora and nominated as a good candidate for paratransgenic control of malaria.Objectives: The aim of this study was evaluate the population dynamics of E. cloacae within An. stephensi and its introduction methods to the mosquitoes Anopheles stephensi in laboratory and semi-field condition.Materials and Methods: Enterobacter cloacae subsp. dissolvens expressing green fluorescent protein and defensin (GFP-D) was used to study transstadial transmission and the course of time, larval habitat, sugar, and blood meal on dynamics of the bacterium in the mosquito life stages. The bacterium fixed on corn-cob formulation (CCF) and released on larval breeding place. Also sugar solution including bacterial suspensions [1 × 109cells/mL], 5% (wt/vol) fructose and red dye (1/50ml) soaked in cotton pads were used for bacterial dynamics in sugar bait and mosquito midgut. The bacterial quantities were measured by plating samples and counting GFP expressing colonies on the Tet-BHI agar medium.Results: The CCF was found suitable to releasing the transgenic bacteria in larval habitats. The E. cloacae population remained stable in sugar bait at least for eleven days in lab condition in comparison with semi-field condition where the increasing trend of bacteria population is perceptible. The bacteria population was decreased in Anopheles larvae over the time and it was strongly dependent with bacterial population in larval habitats. The bacterium was weakly transmitted transstadialy from larval to adult stage. The bacterial population in the sugar-fed female mosquitoes was decreased gradually during 5 days in semi-field condition while it increased smoothly and then dramatically in the An. stephensi gut following sugar and blood meal respectively followed by a gradual reduction over the time in laboratory condition.Conclusion: Enterobacter cloacae was stable in sugar bait and increased tremendously in the gut of female adult An. stephensi within 24h post blood meal. This bacterium could be introduced in the field condition with attractive sugar solution and sugar bait stations in a paratransgenic approach. It is recommended to evaluate the attraction of sugar bait in combination with attractive kairomones as well as its stability and survival rate in the field conditions.

کلیدواژه ها:

Bacterial dynamic ، Enterobacter cloacae ، Paratransgenesis